Methods and compositions for generating an immune response by inducing cd40 and pattern recognition receptors and adaptors thereof

ABSTRACT

Provided are methods for activating an antigen-presenting cell and eliciting an immune response by inducing pattern recognition receptor activity, and CD40 activity. Also provided are methods for activating an antigen-presenting cell and eliciting an immune response by inducing CD40 activity without prostaglandin E2. Also provided are methods for activating an antigen-presenting cell and eliciting an immune response by inducing an inducible chimeric molecule comprising a region of a pattern recognition receptor or an adaptor thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 16/795,154, filed on Feb. 19, 2020, which is a continuation ofU.S. patent application Ser. No. 15/857,265, filed on Dec. 28, 2017 (nowabandoned), which is a continuation of U.S. patent application Ser. No.14/191,167, filed on Feb. 26, 2014 (now abandoned), which is acontinuation of U.S. patent application Ser. No. 12/445,939, filed onOct. 26, 2010 (now U.S. Pat. No. 8,691,210), which is a 371 nationalphase of international patent application number PCT/US2007/081963,filed on Oct. 19, 2007, which claims the benefit of priority to each ofU.S. Provisional Application serial number U.S. 60/895,088, filed Mar.15, 2007, and U.S. Provisional Application serial number U.S.60/862,211, filed Oct. 19, 2006, each of which is incorporated herein byreference, including all text, tables and drawings in their entirety.

SEQUENCE LISTING DISCLOSURE

This application contains a computer readable Sequence Listing which hasbeen submitted in XML file format with this application, the entirecontent of which is incorporated by reference herein in its entirety.The Sequence Listing XML file submitted with this application isentitled “14562-105-999_SEQ_LISTING.xml”, was created on Oct. 20, 2022,and is 45,674 bytes in size.

FIELD OF THE INVENTION

The invention relates generally to the field of immunology, and inparticular, methods and compositions for activating antigen-presentingcells and for inducing immune responses.

BACKGROUND

Due to their unique method of processing and presenting antigens and thepotential for high-level expression of costimulatory and cytokinemolecules, dendritic cells (DC) are effective antigen-presenting cells(APCs) for priming and activating naïve T cells¹. This property has ledto their widespread use as a cellular platform for vaccination in anumber of clinical trials with encouraging results^(2,3). However, theclinical efficacy of DC vaccines in cancer patients has beenunsatisfactory, probably due to a number of key deficiencies, includingsuboptimal activation, limited migration to draining lymph nodes, and aninsufficient life span for optimal T cell activation in the lymph nodeenvironment.

A parameter in the optimization of DC-based cancer vaccines is theinteraction of DCs with immune effector cells, such as CD4+, CD8+ Tcells and T regulatory (Treg) cells. In these interactions, thematuration state of the DCs is a key factor in determining the resultingeffector functions⁴. To maximize CD4+ and CD8+ T cell priming whileminimizing Treg expansion, DCs need to be fully mature, expressing highlevels of co-stimulatory molecules, (like CD40, CD80, and CD86), andpro-inflammatory cytokines, like IL-12p70 and IL-6. Equally important,the DCs must be able to migrate efficiently from the site of vaccinationto draining lymph nodes to initiate T cell interactions⁵.

For the ex vivo maturation of monocyte-derived immature DCs, themajority of DC-based trials have used a standard maturation cytokinecocktail (MC), comprised of TNF-alpha, IL-1beta, IL-6, and PGE₂. Theprincipal function of prostaglandin E2 (PGE₂) in the standard maturationcocktail is to sensitize the CC chemokine receptor 7 (CCR7) to itsligands, CC chemokine ligand 19 (CCL19) and CCL21 and thereby enhancethe migratory capacity of DCs to the draining lymph nodes^(6,7).However, PGE₂ has also been reported to have numerous properties thatare potentially deleterious to the stimulation of an immune response,including suppression of T-cell proliferation,^(8,9) inhibition ofpro-inflammatory cytokine production (e.g., IL-12p70 and TNF-alpha^(10,11)), and down-regulation of major histocompatibility complex (MHC)II surface expression¹². Therefore, maturation protocols that can avoidPGE₂ while promoting migration are likely to improve the therapeuticefficacy of DC-based vaccines.

A DC activation system based on targeted temporal control of the CD40signaling pathway has been developed to extend the pro-stimulatory stateof DCs within lymphoid tissues. DC functionality was improved byincreasing both the amplitude and the duration of CD40 signaling¹³. Toaccomplish this, the CD40 receptor was re-engineered so that thecytoplasmic domain of CD40 was fused to synthetic ligand-binding domainsalong with a membrane-targeting sequence. Administration of alipid-permeable, dimerizing drug, AP20187 (AP), called a chemicalinducer of dimerization (CID)¹⁴, led to the in vivo induction ofCD40-dependent signaling cascades in murine DCs. This induction strategysignificantly enhanced the immunogenicity against both defined antigensand tumors in vivo beyond that achieved with other activationmodalities¹³. The robust potency of this chimeric ligand-inducible CD40(named iCD40) in mice suggested that this method might enhance thepotency of human DC vaccines, as well.

Pattern recognition receptor (PRR) signaling, an example of which isToll-like receptor (TLR) signaling also plays a critical role in theinduction of DC maturation and activation, and human DCs express,multiple distinct TLRs¹⁵. The eleven mammalian TLRs respond to variouspathogen-derived macromolecules, contributing to the activation ofinnate immune responses along with initiation of adaptive immunity.Lipopolysaccharide (LPS) and a clinically relevant derivative,monophosphoryl lipid A (MPL), bind to cell surface TLR-4 complexes¹⁶,leading to various signaling pathways that culminate in the induction oftranscription factors, such as NF-kappaB and IRF3, along withmitogen-activated protein kinases (MAPK) p38 and c-Jun kinase(JNK)^(17,18). During this process DCs mature, and partially upregulatepro-inflammatory cytokines, like IL-6, IL-12, and Type I interferons¹⁹.LPS-induced maturation has been shown to enhance the ability of DCs tostimulate antigen-specific T cell responses in vitro and in vivo²⁶.

SUMMARY

An inducible CD40 (iCD40) system has been applied to human dendriticcells (DCs) and it has been demonstrated that combining iCD40 signalingwith Pattern recognition receptor (PRR) ligation causes persistent androbust activation of human DCs. These activated DCs not only possesshigh migratory capacity in vitro and in vivo, but also produce highlevels of IL-12 and potently activate antigen-specific helper (TH1) andcytotoxic T lymphocytes. These studies demonstrate potent DC activationand migratory capacity can be achieved in the absence of maturationcocktails that contain PGE₂. These features form the basis of cancerimmunotherapies for treating such cancers as advanced,hormone-refractory prostate cancer, for example. Accordingly, it hasbeen discovered that the combination of inducing CD40 and a PRRsynergistically activates antigen-presenting cells and induces an immuneresponse against an antigen. It also has been discovered thatantigen-presenting cells can be activated and immune responses can begenerated against an antigen by inducing CD40.

Thus, provided herein is a method for activating an antigen-presentingcell, which comprises: (a) transducing an antigen-presenting cell with anucleic acid having a nucleotide sequence that encodes a chimericprotein, wherein the chimeric protein comprises a membrane targetingregion, a ligand-binding region and a CD40 cytoplasmic polypeptideregion lacking the CD40 extracellular domain; (b) contacting theantigen-presenting cell with a non-protein multimeric ligand that bindsto the ligand-binding region; and (c) contacting the antigen-presentingcell with a PRR ligand, for example, a TLR ligand whereby theantigen-presenting cell is activated. In certain embodiments,antigen-presenting cell is not contacted with prostaglandin E₂ (PGE₂)when contacted with the multimeric ligand, and in particularembodiments, the antigen-presenting cell is not contacted with acomposition comprising prostaglandin E₂ (PGE₂) and one or morecomponents selected from the group consisting of IL-1beta, IL-6 and TNFalpha.

Also provided is a method for activating an antigen-presenting cell,which comprises: (a) transducing an antigen-presenting cell with anucleic acid having a nucleotide sequence that encodes a chimericprotein, wherein the chimeric protein comprises a membrane targetingregion, a ligand-binding region and a CD40 cytoplasmic polypeptideregion lacking the CD40 extracellular domain; and (b) contacting theantigen-presenting cell with a non-protein multimeric ligand that bindsto the ligand-binding region, wherein the antigen-presenting cell is notcontacted with prostaglandin E2 (PGE₂) when contacted with themultimeric ligand, whereby the antigen-presenting cell is activated. Insome embodiments, the method further comprises contacting theantigen-presenting cell with a PRR ligand, for example, a Toll-likereceptor (TLR) ligand.

Further, provided is a method for inducing a cytotoxic T lymphocyte(CTL) immune response against an antigen, which comprises: contacting anantigen-presenting cell sensitized with an antigen with: (a) amultimeric ligand that binds to a chimeric protein in the cell, whereinthe chimeric protein comprises a membrane targeting region, aligand-binding region and a CD40 cytoplasmic polypeptide region lackingthe CD40 extracellular domain, and (b) a PRR ligand, for example, aToll-like receptor (TLR) ligand; whereby a CTL immune response isinduced against the antigen. In certain embodiments, antigen-presentingcell is not contacted with prostaglandin E₂ (PGE₂) when contacted withthe multimeric ligand, and in particular embodiments, theantigen-presenting cell is not contacted with a composition comprisingprostaglandin E₂ (PGE₂) and one or more components selected from thegroup consisting of IL-1beta, IL-6 and TNF alpha.

Also provided is a method for inducing an immune response against anantigen, which comprises: contacting an antigen-presenting cellsensitized with an antigen with a multmeric ligand that binds to achimeric protein in the cell, wherein: (a) the chimeric proteincomprises a membrane targeting region, a ligand-binding region and aCD40 cytoplasmic polypeptide region lacking the CD40 extracellulardomain, and (b) the antigen-presenting cell is not contacted withprostaglandin E₂ (PGE₂) when contacted with the multimeric ligand;whereby an immune response against the antigen is induced. The methodcan further comprise contacting the antigen-presenting cell with a PRRligand, for example, a TLR ligand.

Provided also is a method for inducing a cytotoxic T lymphocyte (CTL)immune response against an antigen, which comprises: contacting a humanantigen-presenting cell sensitized with an antigen with: (a) amultimeric molecule having two or more regions that bind to andmultimerize native CD40, and (b) a PRR ligand, for example, a TLR ligandligand; whereby a CTL immune response is induced against the antigen. Insuch methods, the multimeric molecule can be an antibody that binds toan epitope in the CD40 extracellular domain (e.g., humanized anti-CD40antibody; Tai et al., Cancer Research 64, 2846-2852 (2004)), can be aCD40 ligand (e.g., U.S. Pat. No. 6,497,876 (Maraskovsky et al.)) or maybe another co-stimulatory molecule (e.g., B7/CD28).

In the methods for inducing an immune response presented herein, theantigen-presenting cell can be transduced ex vivo or in vivo with anucleic acid that encodes the chimeric protein. The antigen-presentingcell may be sensitized to the antigen at the same time theantigen-presenting cell is contacted with the multimeric ligand, or theantigen-presenting cell can be pre-sensitized to the antigen before theantigen-presenting cell is contacted with the multimerization ligand. Insome embodiments, the antigen-presenting cell is contacted with theantigen ex vivo. In certain embodiments the antigen-presenting cell istransduced with the nucleic acid ex vivo and administered to the subjectby intradermal administration, and sometimes the antigen-presenting cellis transduced with the nucleic acid ex vivo and administered to thesubject by subcutaneous administration. The antigen may be a tumorantigen, and the CTL immune response can induced by migration of theantigen-presenting cell to a draining lymph node.

Also provided herein is a composition comprising an antigen-presentingcell and a PRR ligand, for example, a TLR ligand, wherein: theantigen-presenting cell is transduced with a nucleic acid having anucleotide sequence that encodes a chimeric protein, and the chimericprotein comprises a membrane targeting region, a ligand-binding regionand a CD40 cytoplasmic polypeptide region lacking the CD40 extracellulardomain. The composition may further comprise a non-protein multimericligand that binds to the ligand-binding region.

In the methods and compositions presented herein, the membrane targetingregion can be a myristoylation targeting region. In some embodiments,the CD40 cytoplasmic polypeptide region is encoded by a polynucleotidesequence in SEQ ID NO: 1. The multimeric ligand often is a smallmolecule and it sometimes is dimeric, such as a dimeric FK506 or adimeric FK506 analog (e.g., AP1903). Any suitable PRR ligand, forexample, any suitable TLR ligand can be utilized, and can be selected bythe person of ordinary skill in the art (e.g., Napolitani et al., NatureImmunology, Advanced Online Publication doi:10.1038/ni 1223 (2005)). TheTLR ligand in certain embodiments is selected from the group consistingof lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), FSL-1, Pam3,CSK4, poly(I:C), synthetic imidazoquinoline resiquimod (R848; U.S. Pat.No. 6,558,951 to Tomai et al.) and CpG, and the TLR ligand sometimes isa TLR4 ligand such as LPS or MPL. The nucleic acid can be containedwithin a viral vector, such as an adenoviral vector, for example. Incertain embodiments, the antigen-presenting cell is transduced with thenucleic acid ex vivo or in vivo, and sometimes the antigen-presentingcell is a dendritic cell, such as a human dendritic cell, for example.

Also provided herein is a method for assessing migration of anantigen-presenting cell to a lymph node, which comprises: (a) injectinginto a subject an antigen-presenting cell that produces a detectableprotein, and (b) determining the amount of the detectable protein in thelymph node of the animal, whereby migration of the antigen-presentingcell to the lymph node is assessed from the amount of the detectableprotein in the lymph node. In such methods the animal can be a rodent,such as a rat or a mouse (e.g., irradiated mouse). In some embodiments,the detectable protein is a luciferase protein, such as a chick beetle(e.g., Pyrophorus plagiophalamus) red-shifted luciferase protein. Incertain embodiments, the antigen-presenting cell has been transducedwith a nucleic acid having a polynucleotide sequence that encodes thedetectable protein. In certain embodiments, the lymph node is thepopliteal lymph node or inguinal lymph node. The antigen-presenting cellcan be a dendritic cell, such as a human dendritic cell. In certainembodiments, the lymph node is removed from the animal before the amountof detectable protein is determined, and sometimes the D-Luciferin isadministered to the removed lymph node. The amount of the detectableprotein may be qualitative (e.g., relative amounts compared acrossdifferent samples) and can be quantitative (e.g., a concentration). Theamount of the detectable protein may be determined by directly detectingthe protein. For example, the protein may be fluorescent (e.g., greenfluorescent protein or a red-shifted or blue-shifted version) or can bebound to a fluorescent label (e.g., an antibody linked to afluorophore). Alternatively, the amount of the detectable protein candetermined indirectly by administering a substrate to the animal that isconverted into a detectable product by the protein and detecting thedetectable product. For example, the amount of a luciferase protein canbe determined by administering D-Luciferin to the animal and detectingthe D-Luciferin product generated by the luciferase produced in theantigen-presenting cell.

Provided also in the present invention are methods for activating anantigen-presenting cell, which comprise: transducing (or transfecting)an antigen-presenting cell with a nucleic acid having a nucleotidesequence that encodes a chimeric protein, wherein the chimeric proteincomprises (i) a membrane-targeting region, (ii) a ligand-binding regionand (iii-a) a signaling region and/or cytoplasmic region of a patternrecognition receptor (PRR) or (iii-b) an adapter of a PRR; andcontacting the antigen-presenting cell with a non-protein multimericligand that binds to the ligand-binding region; whereby theantigen-presenting cell is activated. In certain embodiments thechimeric protein comprises a CD40 cytoplasmic polypeptide region lackingthe CD40 extracellular domain. The CD40 cytoplasmic polypeptide regionin certain embodiments is encoded by a polynucleotide sequence in SEQ IDNO: 1

In some embodiments the chimeric protein comprises a signaling regionand/or cytoplasmic region of a PRR. Sometimes the PRR is a NOD-like PRR,such as a NOD1 PRR or a NOD2 PRR, for example. In certain embodimentsthe PRR is not a NOD-like PRR, and is not a NOD1 PRR or a NOD2 PRR, forexample. The PRR in some embodiments is a RIG-like helicase (RLH), suchas a RIG-I PRR or an Mda-5 PRR, for example. The PRR sometimes is aToll-like receptor (TLR) PRR, such as a TLR3, TLR4, TLR7, TLR8 and TLR9,and in certain embodiments the chimeric protein comprises a cytoplasmicregion, or a TIR (Toll/IL-1R) region, from a TLR PRR. In certainembodiments the chimeric protein comprises an adapter that binds to aPRR of any one of embodiments described herein. The adaptor may beselected from the group consisting of MyD88, TRIF/TICAM-1, TIRAM/ICAM-2,MAL/TIRAP, or protein-protein interaction domains from said adaptors,such as TIR, CARD or pyrin domains (PYD) in certain non-limitingembodiments. Nucleic acid sequences and protein sequences of suchmolecules, and signaling regions and cytoplasmic regions therein, areknown to the person of ordinary skill in the art (e.g., World Wide Webaddress ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene).

In certain embodiments, the membrane targeting region is amyristoylation-targeting region, although the membrane-targeting regioncan be selected from other types of transmembrane-targeting regions,such as regions described hereafter. In some embodiments the ligand is asmall molecule, and sometimes the molecule is dimeric. Examples ofdimeric molecules are dimeric FK506 and dimeric FK506 analogs. Incertain embodiments the ligand is AP1903 or AP20187. In someembodiments, the chimeric protein includes one or more ligand-bindingregions, such as two or three ligand-binding regions, for example. Theligand-binding regions often are tandem.

The nucleic acid in certain embodiments is contained within a viralvector, such as an adenoviral vector for example. The antigen-presentingcell in some embodiments is contacted with an antigen, sometimes exvivo. In certain embodiments the antigen-presenting cell is in a subjectand an immune response is generated against the antigen, such as acytotoxic T-lymphocyte (CTL) immune response. In certain embodiments, animmune response is generated against a tumor antigen (e.g., PSMA). Insome embodiments, the nucleic acid is prepared ex vivo and administeredto the subject by intradermal administration or by subcutaneousadministration, for example. Sometimes the antigen-presenting cell istransduced or transfected with the nucleic acid ex vivo or in vivo. Insome embodiments, the nucleic acid comprises a promoter sequenceoperably linked to the polynucleotide sequence. Alternatively, thenucleic acid comprises an ex vivo-transcribed RNA, containing theprotein-coding region of the chimeric protein.

Also provided herein is a composition which comprises a nucleic acidhaving a polynucleotide sequence that encodes a chimeric protein,wherein the chimeric protein comprises (i) a membrane targeting region,(ii) a ligand-binding region that binds to a multimeric non-proteinligand, and (iii-a) a signaling region and/or cytoplasmic region of apattern recognition receptor (PRR) or (iii-b) an adapter of a PRR.Embodiments pertaining to methods described above also are applicable tocompositions herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Schematic diagram of iCD40 and expression in human DCs.FIG. 1A. The human CD40 cytoplasmic domain can be subcloned downstreamof a myristoylation-targeting domain (M) and two tandem domains (Fv)22.The expression of M-Fv-Fv-CD40 chimeric protein, referred to here asinducible CD40 (iCD40) can be under cytomegalovirus (CMV) promotercontrol. FIG. 1B. The expression of endogenous (eCD40) and recombinantinducible (iCD40) forms of CD40 assessed by Western blot. Lane 1, wildtype DCs (endogenous CD40 control); lane 2, DCs stimulated with 1microgram/ml of LPS; lanes 3 and 4, DCs transduced with 10,000 VP/cell(MOI˜160) of Ad5/f35-iCD40 (iCD40-DCs) with and without AP20187dimerizer drug respectively; lane 5, iCD40-DCs stimulated with LPS andAP20187; lane 6, DCs stimulated with CD40L and LPS; lane 7, DCstransduced with Ad5/f35-GFP (GFP-DCs) at MOI 160 and stimulated withAP20187 and LPS; lane 8, GFP-DCs stimulated with AP20187; lane 9, 293 Tcells transduced with Ad5/f35-iCD40 (positive control for inducible formof CD40). The expression levels of alpha-tubulin served as internalcontrol.

FIG. 2 Enhanced maturation status of iCD40 DCs stimulated with LPS.Immature DCs were transduced with Ad5/f35-iCD40 or Ad5/f35-Luciferase(Luc) and stimulated with LPS (1 microgram/ml) for 48 hours in presenceof 100 nM AP20187 (AP). Alternatively, DCs were matured in the presenceof LPS alone. Percentage of DCs expressing CD40, CD80, CD83 and CD86 wasdetermined using PE-conjugated anti-human mAbs (BD Biosciences) by flowcytometric analysis. FACS histograms from one donor (out of at leastfive) experiment are shown.

FIGS. 3A-3E. Synergism of iCD40 and TLR-4 for IL-12p70 and IL-6production. FIG. 3A. Immature human DCs (5×105) were transduced withAd5/f35-iCD40 or Ad5/f35-Luciferase (Luc) and stimulated with 1microgram/ml of LPS or MPL for 48 hours in the presence of 100 nMAP20187 (AP) dimerizer drug. Alternatively, DCs were stimulated withstandard maturation cocktail (MC), or with MC lacking PGE₂ (MC w/oPGE₂). The supernatants were assayed by ELISA (in duplicate) forIL-12p70 level 48 hours following various treatments. FIG. 3B. DCs weretransduced with Ad5/f35-iCD40 and stimulated with FSL-1, Pam3CSK4 or MPLfor 48 hours. The supernatants were assayed by ELISA 48 hourspost-stimulation. FIG. 3C. DCs were transduced with Ad5/f35-iCD40 andstimulated with 1 microgram/ml LPS or CD40L (Alexis Biochemicals).IL-12p70 production was monitored at 6 h, 12 h, 18 h, 24 h, 48 h, 72 hand 96 h post-stimulation (left panel). In parallel, cells of the samedonor were washed 3 times 24 h post-stimulation and cultured in mediumwithout stimulatory factors. The expression of IL-12p70 was monitoredfor 3 more days, every 24 hours (right panel). FIG. 3D. Expression ofhuman SOCS1 gene was measured in DCs transduced and stimulated (asdescribed above) for 24 hours. The expression levels were measured induplicates and normalized by 18S ribosomal RNA housekeeping geneexpression. The fold increase above mock expression is shown. FIG. 3E.DCs were transduced with Ad5/f35-iCD40 or Ad5/f35-Luc and stimulatedwith LPS, MPL and CD40L for 48 hours with or without 100 nM of AP20187dimerizer drug. The supernatants were assayed by ELISA (in duplicates)for IL-6 48 h following various treatments. All the experiments wereperformed with DCs from at least three different donors. The IL-12p70and IL-6 expression levels were measured from at least 5 differentdonors.

FIGS. 4A and 4B. iCD40-DCs significantly induce antigen-specific TH1polarized CD4+ T cells. FIG. 4A. DCs from HLA DR11.5 donor were pulsedwith tetanus toxoid and transduced with the described agents. AutologousCD4+CD45RA+ T cells were co-cultured with DCs (at DC T cell ratio 1:10)for 7 days and restimulated at day 8 with DCs pulsed with TTp30 peptide(FNNFTVSFWLRVPKVSASHLE) (SEQ ID NO: 5). T cells were double stained withanti-interferon-gamma-FITC and anti-CD4-PE antibodies. The percentage ofCD4+/IFN-gamma+ T cells is indicated. FIG. 4B. Supernatants wereharvested and analyzed by BD Cytometric Bead Array Flex Set forexpression of IFN-gamma, TNF-alpha, IL-4, and IL-5. Results of oneexperiment out of three are shown.

FIGS. 5A-5C. Enhanced induction of MAGE-3 antigen-specific CTL byiCD40-DCs. DCs derived from HLA-A2 positive donors were transduced withindicated reagents and pulsed with 25 micrograms/ml of MAGE3 protein.DCs were cultured with autologous T-cells (1:3 DC:T cell ratio) for 7days in complete RPMI supplemented with 20 IU/ml of hIL-2. T cells wererestimulated with DCs at day 7. FIG. 5A. Frequency of MAGE3 2.1peptide-specific T cells were determined by IFN-gamma ELISPOT analysis.100,000 T cells/well were stimulated with MAGE3 2.1 or GAG 2.1 (negativecontrol)/irrelevant peptide) or cultured without stimulation (mock).FIG. 5B. DCs from HLA-A2 positive donor were co-cultured with autologousT cells. After three serial stimulations with DCs, T cells wereevaluated for antigen-specific lytic activity using a 51Cr releaseassay. The assays were performed in triplicate. IM, influenza matrixpeptide. FIG. 5C. The effector T cell populations generated after serialstimulation with DCs were stained with MAGE3 A2.1 peptide-loaded HLA-A2tetramer. MAGE3 peptide-specific CD8+ T cells were identified using flowcytometry. The percentages indicate the fraction of tetramer-positivecells within the entire populations of CD8+ T cells. Representativeresults of one experiment out of three (independent donors) performedare shown.

FIGS. 6A and 6B. Enhanced cytolytic function of PSMA-specific CTLinduced by iCD40-DCs. FIG. 6A. DCs generated from HLA-A2+ malevolunteers were pulsed with 50 micrograms/ml PSMA protein, transducedwith Ad-iCD40 or Ad-Luc and cultured with LPS (1 micrograms/ml) or MC.Antigen-specific CU activity was assessed by chromium-release assay.FIG. 6B. DCs of the same HLA-A2+ male donor were pulsed with MAGE-3 2.1peptide. MAGE-3-specific CTL cytolytic activity was measured inchromium-release assay.

FIGS. 7A-7D. Up-regulation of CCR7 expression and enhanced migratorycapacities of iCD40-DCs. FIG. 7A. Human DCs were transduced withAd-iCD40 (iCD40) or Ad-Luc (Luc) and cultured for 48 h with 100 nMAP20187 (AP), MC and 1 micrograms/ml LPS or MPL. CCR7 expression wasmeasured using PE-conjugated anti-human CCR7 mAb. The percentage ofCCR7-positive cells is indicated. Similar results were obtained for atleast five different donors. FIG. 7B. Human DCs were transduced with10,000 VP/cell of Ad5f35-iCD40 (iCD40) or Ad-Luciferase (Luc) andincubated for 48 hours with 1 micrograms/ml MPL or MC and 100 nMAP20187. DCs were labeled with Green-CMFDA cell tracker and added to theupper chamber. Fluorescence of cells migrated through the microporousmembrane was measured. Each experiment (including the controlspontaneous migration to the medium) was performed in triplicate for atleast four different donors. FIG. 7C. Human DCs were transduced withAd-CBR-Luc or iCD40 and stimulated as indicated. Mouse DCs (mDCs) weretransduced with Ad-CBR-Luc and stimulated with LPS. 2×106 DCs wereinjected into both hind footpads of three mice/group (n=6). Mice wereimaged at day 2 after inoculation (upper panel), and popliteal lymphnodes (lower panel) were removed at day 2 post-DC inoculation. FIG. 7D.Mean luminescent signal from the removed popliteal and inguinal LNs wasmeasured and normalized by background subtraction (* p<0.05, *** p<0.001compared to mock DCs).

FIG. 8 is a supplementary figure to FIG. 1 .

FIG. 9 is a supplementary figure to FIG. 2 .

FIG. 10 . Schematic of iCD40. Administration of the lipid-permeabledimerizing drug, AP20187/AP19031, leads to oligomerization of thecytoplasmic domain of CD40, modified to contain AP20187-binding domainsand a myristoylation-targeting sequence.

FIGS. 11A-11F. iCD40 activates primary DCs and prolongs their longevity.FIG. 11A. Western blot (

-HA) of primary DCs infected with AD-iCD40-GFP. FIG. 11B. Flow cytometryanalysis of transduced DCs. FIG. 11C. Flow cytometry of Kb, B7.2 andendogenous CD40 on iCD40-stimulated DCs. FIG. 11D. Kinetics of IL-12induction (ELISA) by iCD40 and LPS. FIG. 11E. Survival kinetics of DCsfollowing CD40L or iCD40 stimulation. FIG. 11F. Survival kinetic of DCsin vivo. Draining popliteal lymph nodes were collected 42 h after DCinjection, and propidium iodide-negative populations were analyzed byflow cytometry.

FIGS. 12A-12C. iCD40 enhances the efficacy of DC-based tumor vaccinesand the potency of DC-mediated tumor immunosurveillance. FIG. 12A.Activation of SIINFEKL-pulsed (SEQ ID NO: 6) iCD40 BMDCs with LPS orCD40L or both in vitro, or with CD40-specific mAb in vivo, show noefficacy towards large (greater than 0.5 cm3) EG.7-OVA tumors. Opensquare, PBS.exp.1; filled triangle, PBS.exp.2; open inverted triangle,DC; open diamond, DC+LPS; open circle, DC+LPS/CD40L; filled square,DC+rat IgG; open triangle, CD40-specific mAb in vivo. FIG. 12B. In vivodrug-mediated activation of iCD40-expressing DCs eliminates establishedEG.7-OVA tumors after a single vaccination. Filled square, PBS; opentriangle, iCD40 DC; open inverted triangle, iCD40 DC+AP20187 in vitro;open diamond, iCD40. FIG. 12C. To confirm the elicitation of tumorantigen-specific T cell responses in tumor-bearing mice, H-2KbOVA257-264 tetramer analysis was performed on peripheral blood CD8+ Tcells.

FIGS. 13A-13E. MF-

Akt and M-Akt induce BMDC longevity in vitro and in vivo. FIGS. 13A and13B. BMDCs were left untreated (

), or treated with LPS (

), Ad-EGFP (

) or Ad-M-Akt (

) at 100 m.o.i. and further incubated for 2 to 5 d without GM-CSF. Invitro DC apoptosis examined by Annexin V-PE staining. Histograms of d5(thinner line) were compared to that of d2 (thicker) FIG. 13A, Errorbars=mean+std. of results pooled from three independent experiments. *,P<0.05 between Ad-EGFP and Ad-M-Akt FIG. 13B. FIGS. 13C-13E. Effect ofAd-MF-

Akt on BMDC longevity, in vivo. CFSE-stained BMDCs were untreated (

), or treated with LPS (

), Ad-EGFP (

) or Ad-MF-ΔAkt (

) for 2 hr before injection into hind legs of syngeneic mice (n=2-4 pertime point). FIG. 13C. After indicated times, draining popliteal LNcells were stained with PI. PI−/CFSE+ cells were analyzed by flowcytometry. Background CFSE+ from PBS control (−) was subtracted for eachvalue. FIG. 13D. Boxed numbers indicate d 5 CFSE+ percent. FIG. 13E.Representative LNs isolated from indicated mice on days 7 and 10.

FIGS. 14A-14C. MF-

Akt expression enhances the efficacy of DC-based tumor vaccines. FIG.14A. Syngeneic BL/6 mice (n=5) challenged with EG.7-ova cells (2×106) atd0 were treated with PBS (

) or 2×106 BMDCs (

) pulsed with SIINFEKL (SEQ ID NO: 6) peptide (10

g/ml) and LPS (1

g/ml) (

), 100 m.o.i. of Ad-EGFP (

) or Ad-MF-

Akt (

) at d7, and tumor sizes were recorded biweekly. Numbers indicatefraction of mice bearing tumors (>0.1 cm3). *, P<0.05. FIG. 14B.Representative examples of EG7-OVA tumor-bearing mice vaccinated withAd-EGFP or Ad-MF-

Akt BMDCs. Tumors were compared on d7 and d14 after vaccination. FIG.14C. PBMCs from indicated group at d21 were isolated and stained withPE-KbSIINFEKL (SEQ ID NO: 6) tetramer and FITC-conjugated CD8. FIG. 14D.Mean percentage of CD8+ and KbSIINFEKL (SEQ ID NO: 6) tetramer positivepopulation in PBMCs from two to three mice per group. Error barsrepresent mean±S.E.M. *, p<0.05, **, P<0.005.

FIG. 15 is a schematic of CID-inducible TLRs.

FIG. 16 Inducible TLR7 and 8 signal in Jurkat T cells. Jurkat TAg cellswere co-transiently transfected with NF-kappaB-SEAP reporter plasmidalong with various iTLRs, positive control ihCD40, or negative controlvector. After 24 h cells were treated with dilutions of CID for anadditional 20 hrs. Average of 2 wells shown. Representative of 3experiments. All constructs verified by sequence and protein analysis.

FIG. 17 . Detection of chemiluminescent B16 tumors in syngeneic C57BL/6mice. B16 melanoma cells were stably transfected with expressionplasmid, pEF1

-CBR-IRES-Neo. 10E5 cells were injected subQ and imaged using an IVIS™imaging system 5 days later following i.p. D-Luciferin (˜1 mg)injection. Note: even non-palpable tumors detected.

FIG. 18 is a schematic from Malissen & Ewbank (05) Nat. Imm. 6, 749.

FIG. 19 is a schematic of CID-inducible composite Toll-like receptors(icTLRs).

FIG. 20 is a schematic of CID-inducible composite TLR (icTLRs)/CD40.

FIG. 21 . Synergism of TLR4 and iCD40 signaling. MoDCs were stained forCD83 expression 48 h following MPL (1 mg/ml), MC (IL1b (150 ng/ml), IL6(150 ng/ml), TNFa (10 ng/ml), PGE2 (1 mg/ml)), iCD40 (10 kvp/cell)/AP20187 (100 nM)+MPL, or mock stimulation. Percentage CD83+cells shown.

FIG. 22 . Synergism of TLR4 and iCD40 signaling for IL12p70 production.Supernatants were assayed by ELISA for IL12p70 levels following varioustreatments (48 hrs) of MoDCs. In this experiment only iCD40+ TLR4ligation (with MPL or LPS) led to high-level IL12 production.

FIG. 23 . Inducible CD40 triggers migration as well as standardmaturation cocktail (MC). MoDCs were transduced with 10 k vp/cellAd5/f35-ihCD40 (iCD40) or Ad5/f35-GFP (GFP), treated (48 h) with AP20187(CID), MC (±PGE2), MPL or nothing and were labeled withmembrane-impermeant fluorescent dye, Green-CMFDA. 5000 cells were placedin the top chamber of a 96-well HTS Fluoroblok plates (BD Falcon) andspecific migration (in 30′) across an 8

m filter to the lower chamber containing CCL19 (100 ng/ml) was measuredby a FLUOstar OPTIMA reader (BMG Labtech, Inc.) at 485/520 nm andsubtracted from background migration. Representative of at least 5 expsperformed in triplicate.

FIG. 24 : The principal relationships between the Toll-like receptors(TLRs), their adaptors, protein kinases that are linked to them, anddownstream signaling effects. Nature 430, 257-263(8 Jul. 2004).

FIG. 25A. Chimeric iTLR4s in RAW 264.7 cells

RAW 264.7 cells were cotransfected transiently with 3 microgramexpression plasmids for chimeric iTLR4s and 1 microgramNFkappaB-dependent SEAP reporter plasmid (indicated as R in Figure).

FIG. 25B. Chimeric iTLRs in RAW 264.7 cells

RAW 264.7 cells were cotransfected transiently with 3 microgramexpression plasmids for chimeric iTLRs and 1 microgram SEAP reporterplasmid. iTLR4, iTLR7 and iCD40 activity were tested using aNF-kappaB-dependent reporter while iTLR3 activity was tested using anIFNgamma-dependent reporter plasmid. iCD40 was used as the positivecontrol. 1 microgram/ml LPS was used as a positive control for reporteractivity.

FIG. 26 . iNod2 and iCD40 in 293 cells

293 cells were cotransfected transiently at the rate of 1 millioncells/well (of a 6-well plate) with 3 microgram expression plasmids forchimeric iNod-2 and 1 microgram NFkappaB-dependent SEAP reporter plasmid(indicated as R in Figure). iCD40 was used as the positive control.

FIG. 27 . iRIG-1 and iMyD88 in RAW264.7 cells

RAW 264.7 cells were cotransfected transiently with 3 microgramsexpression plasmids for iRIG-1 and 1 micrograms IFNgamma-dependent SEAPreporter plasmid; and 3 micrograms iMyD88 with 1 microgramsNF-kappaB-dependent SEAP reporter plasmid.

FIG. 28 . Schematic of Pattern recognition receptors

FIG. 29 presents an embodiment of an inducible PRR, where 2-3 FKBP12(V36) domains are attached to the amino or carboxy termini of theconserved cytoplasmic signaling domains (T1R) of the representative TLR.The chimeric protein is attached to the plasma membrane with amyristoylation signaling domain or the transmembrane domain of therepresentative TLR.

FIG. 30A. iPRR plasmid embodiments.

FIG. 30B. iPRR plasmid embodiments.

FIG. 30C. iPRR plasmid embodiment.

FIG. 31 is a graph of induction of NF-kappa B SEAP reporter in iRIG,iNOD2, and iCD40-transfected 293 cells.

FIG. 32 is a graph of induction of NF-kappa B SEAP reporter in iRIG-Iand iCD40 transfected 293 cells.

FIG. 33 is a graph of induction of NF-kappa B SEAP reporter in iRIG,iCD40), and iRIG+CD40 transfected 293 cells.

FIG. 34 is a graph of induction of NF-kappa B SEAP reporter in iRIG-Iand iCD40 transfected Jurkat Tag cells.

FIGS. 35A and 35B provide plasmid maps for pSH1-Sn-RIGI-Fy′-Fvls-E andpSH1-Sn-Fv′-Fvls-RIGI-E, respectively. The term “Sn” represents “S” witha NcoI site, added for cloning purposes. The term “S” represents theterm non-targeted.

DETAILED DESCRIPTION

I. Definitions

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Still further, the terms“having”, “including”, “containing” and “comprising” are interchangeableand one of skill in the art is cognizant that these terms are open endedterms.

The term “allogeneic” as used herein, refers to cell types or tissuesthat are antigenically distinct. Thus, cells or tissue transferred fromthe same species can be antigenically distinct.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically competentcells, or both. An antigen can be derived from organisms, subunits ofproteins/antigens, killed or inactivated whole cells or lysates.Exemplary organisms include but are not limited to, Helicobacters,Campylobacters, Clostridia, Corynebacterium diphtheriae, Bordetellapertussis, influenza virus, parainfluenza viruses, respiratory syncytialvirus, Borrelia burgdorferi, Plasmodium, herpes simplex viruses, humanimmunodeficiency virus, papillomavirus, Vibrio cholera, E. coli, measlesvirus, rotavirus, shigella, Salmonella typhi, Neisseria gonorrhea.Therefore, a skilled artisan realizes that any macromolecule, includingvirtually all proteins or peptides, can serve as antigens. Furthermore,antigens can be derived from recombinant or genomic DNA. A skilledartisan realizes that any DNA, which contains nucleotide sequences orpartial nucleotide sequences of a pathogenic genome or a gene or afragment of a gene for a protein that elicits an immune response resultsin synthesis of an antigen. Furthermore, one skilled in the art realizesthat the present invention is not limited to the use of the entirenucleic acid sequence of a gene or genome. It is readily inherent thatthe present invention includes, but is not limited to, the use ofpartial nucleic acid sequences of more than one gene or genome and thatthese nucleic acid sequences are arranged in various combinations toelicit the desired immune response.

The term “antigen-presenting cell” is any of a variety of cells capableof displaying, acquiring, or presenting at least one antigen orantigenic fragment on (or at) its cell surface. In general, the term“antigen-presenting cell” can be any cell that accomplishes the goal ofthe invention by aiding the enhancement of an immune response (i.e.,from the T-cell or —B-cell arms of the immune system) against an antigenor antigenic composition. Such cells can be defined by those of skill inthe art, using methods disclosed herein and in the art. As is understoodby one of ordinary skill in the art (see, for example Kuby, 2000,incorporated herein by reference), and used herein certain embodiments,a cell that displays or presents an antigen normally or preferentiallywith a class II major histocompatibility molecule or complex to animmune cell is an “antigen-presenting cell.” In certain aspects, a cell(e.g., an APC cell) may be fused with another cell, such as arecombinant cell or a tumor cell that expresses the desired antigen.Methods for preparing a fusion of two or more cells is well known in theart, such as for example, the methods disclosed in Goding, pp. 65-66,71-74 1986; Campbell, pp. 75-83, 1984; Kohler and Milstein, 1975; Kohlerand Milstein, 1976, Gefter et al., 1977, each incorporated herein byreference. In some cases, the immune cell to which an antigen-presentingcell displays or presents an antigen to is a CD4+ TH cell. Additionalmolecules expressed on the APC or other immune cells may aid or improvethe enhancement of an immune response. Secreted or soluble molecules,such as for example, cytokines and adjuvants, may also aid or enhancethe immune response against an antigen. Such molecules are well known toone of skill in the art, and various examples are described herein.

The term “cancer” as used herein is defined as a hyperproliferation ofcells whose unique trait—loss of normal controls—results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis.Examples include but are not limited to, melanoma, non-small cell lung,small-cell lung, lung, hepatocarcinoma, leukemia, retinoblastoma,astrocytoma, glioblastoma, gum, tongue, neuroblastoma, head, neck,breast, pancreatic, prostate, renal, bone, testicular, ovarian,mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon,sarcoma or bladder.

The terms “cell,” “cell line,” and “cell culture” as used herein may beused interchangeably. All of these terms also include their progeny,which are any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.

As used herein, the term “iCD40 molecule” is defined as an inducibleCD40. This iCD40 can bypass mechanisms that extinguish endogenous CD40signaling. The term “iCD40” embraces “iCD40 nucleic acids”, “iCD40polypeptides” and/or iCD40 expression vectors. Yet further, it isunderstood the activity of iCD40 as used herein is driven by CID.

As used herein, the term “cDNA” is intended to refer to DNA preparedusing messenger RNA (mRNA) as template. The advantage of using a cDNA,as opposed to genomic DNA or DNA polymerized from a genomic, non- orpartially-processed RNA template, is that the cDNA primarily containscoding sequences of the corresponding protein. There are times when thefull or partial genomic sequence is preferred, such as where thenon-coding regions are required for optimal expression or wherenon-coding regions such as introns are to be targeted in an antisensestrategy.

The term ““dendritic cell” (DC) is an antigen-presenting cell existingin vivo, in vitro, ex vivo, or in a host or subject, or which can bederived from a hematopoietic stem cell or a monocyte. Dendritic cellsand their precursors can be isolated from a variety of lymphoid organs,e.g., spleen, lymph nodes, as well as from bone marrow and peripheralblood. The DC has a characteristic morphology with thin sheets(lamellipodia) extending in multiple directions away from the dendriticcell body. Typically, dendritic cells express high levels of MHC andcostimulatory (e.g., B7-1 and B7-2) molecules. Dendritic cells caninduce antigen specific differentiation of T cells in vitro, and areable to initiate primary T cell responses in vitro and in vivo.

As used herein, the term “expression construct” or “transgene” isdefined as any type of genetic construct containing a nucleic acidcoding for gene products in which part or all of the nucleic acidencoding sequence is capable of being transcribed can be inserted intothe vector. The transcript is translated into a protein, but it need notbe. In certain embodiments, expression includes both transcription of agene and translation of mRNA into a gene product. In other embodiments,expression only includes transcription of the nucleic acid encodinggenes of interest. In the present invention, the term “therapeuticconstruct” may also be used to refer to the expression construct ortransgene. One skilled in the art realizes that the present inventionutilizes the expression construct or transgene as a therapy to treathyperproliferative diseases or disorders, such as cancer, thus theexpression construct or transgene is a therapeutic construct or aprophylactic construct.

As used herein, the term “expression vector” refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules or ribozymes. Expression vectors can contain avariety of control sequences, which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well and are described infra.

As used herein, the term “ex vivo” refers to “outside” the body. One ofskill in the art is aware that ex vivo and in vitro can be usedinterchangeably.

As used herein, the term “functionally equivalent”, as used herein,refers to a CD40 nucleic acid fragment, variant, or analog, refers to anucleic acid that codes for a CD40 polypeptide, or a CD40 polypeptide,that stimulates an immune response to destroy tumors orhyperproliferative disease. Preferably “functionally equivalent” refersto a CD40 polypeptide that is lacking the extracellular domain, but iscapable of amplifying the T cell-mediated tumor killing response byupregulating dendritic cell expression of antigen presentationmolecules.

The term “hyperproliferative disease” is defined as a disease thatresults from a hyperproliferation of cells. Exemplary hyperproliferativediseases include, but are not limited to cancer or autoimmune diseases.Other hyperproliferative diseases may include vascular occulsion,restenosis, atherosclerosis, or inflammatory bowel disease.

As used herein, the term “gene” is defined as a functional protein,polypeptide, or peptide-encoding unit. As will be understood by those inthe art, this functional term includes genomic sequences, cDNAsequences, and smaller engineered gene segments that express, or isadapted to express, proteins, polypeptides, domains, peptides, fusionproteins, and mutants.

The term “immunogenic composition” or “immunogen” refers to a substancethat is capable of provoking an immune response. Examples of immunogensinclude, e.g., antigens, autoantigens that play a role in induction ofautoimmune diseases, and tumor-associated antigens expressed on cancercells.

The term “immunocompromised” as used herein is defined as a subject thathas reduced or weakened immune system. The immunocompromised conditionmay be due to a defect or dysfunction of the immune system or to otherfactors that heighten susceptibility to infection and/or disease.Although such a categorization allows a conceptual basis for evaluation,immunocompromised individuals often do not fit completely into one groupor the other. More than one defect in the body's defense mechanisms maybe affected. For example, individuals with a specific T-lymphocytedefect caused by HIV may also have neutropenia caused by drugs used forantiviral therapy or be immunocompromised because of a breach of theintegrity of the skin and mucous membranes. An immunocompromised statecan result from indwelling central lines or other types of impairmentdue to intravenous drug abuse; or be caused by secondary malignancy,malnutrition, or having been infected with other infectious agents suchas tuberculosis or sexually transmitted diseases, e.g., syphilis orhepatitis.

As used herein, the term “pharmaceutically or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

As used herein, the term “polynucleotide” is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means. Furthermore, one skilled in the art iscognizant that polynucleotides include mutations of the polynucleotides,include but are not limited to, mutation of the nucleotides, ornucleosides by methods well known in the art.

As used herein, the term “polypeptide” is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is interchangeable with the terms “peptides” and“proteins”.

As used herein, the term “promoter” is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa gene.

As used herein, the term “regulate an immune response” or “modulate animmune response” refers to the ability to modify the immune response.For example, the composition of the present invention is capable ofenhancing and/or activating the immune response. Still further, thecomposition of the present invention is also capable of inhibiting theimmune response. The form of regulation is determined by the ligand thatis used with the composition of the present invention. For example, adimeric analog of the chemical results in dimerization of theco-stimulatory polypeptide leading to activation of the DCs, however, amonomeric analog of the chemical does not result in dimerization of theco-stimulatory polypeptide, which would not activate the DCs.

The term “transfection” and “transduction” are interchangeable and referto the process by which an exogenous DNA sequence is introduced into aeukaryotic host cell. Transfection (or transduction) can be achieved byany one of a number of means including electroporation, microinjection,gene gun delivery, retroviral infection, lipofection, superfection andthe like.

As used herein, the term “syngeneic” refers to cells, tissues or animalsthat have genotypes. For example, identical twins or animals of the sameinbred strain. Syngeneic and isogeneic can be used interchangeable.

The term “subject” as used herein includes, but is not limited to, anorganism or animal; a mammal, including, e.g., a human, non-humanprimate (e.g., monkey), mouse, pig, cow, goat, rabbit, rat, guinea pig,hamster, horse, monkey, sheep, or other non-human mammal; a non-mammal,including, e.g., a non-mammalian vertebrate, such as a bird (e.g., achicken or duck) or a fish, and a non-mammalian invertebrate.

As used herein, the term “under transcriptional control” or “operativelylinked” is defined as the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene.

As used herein, the terms “treatment”, “treat”, “treated”, or “treating”refer to prophylaxis and/or therapy. When used with respect to aninfectious disease, for example, the term refers to a prophylactictreatment which increases the resistance of a subject to infection witha pathogen or, in other words, decreases the likelihood that the subjectwill become infected with the pathogen or will show signs of illnessattributable to the infection, as well as a treatment after the subjecthas become infected in order to fight the infection, e. g., reduce oreliminate the infection or prevent it from becoming worse.

As used herein, the term “vaccine” refers to a formulation whichcontains the composition of the present invention and which is in a formthat is capable of being administered to an animal. Typically, thevaccine comprises a conventional saline or buffered aqueous solutionmedium in which the composition of the present invention is suspended ordissolved. In this form, the composition of the present invention can beused conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a subject, the vaccine is able toprovoke an immune response including, but not limited to, the productionof antibodies, cytokines and/or other cellular responses.

II. Dendritic Cells

The innate immune system uses a set of germline-encoded receptors forthe recognition of conserved molecular patterns present inmicroorganisms. These molecular patterns occur in certain constituentsof microorganisms including: lipopolysaccharides, peptidoglycans,lipoteichoic acids, phosphatidyl cholines, bacteria-specific proteins,including lipoproteins, bacterial DNAs, viral single and double-strandedRNAs, unmethylated CpG-DNAs, mannans and a variety of other bacterialand fungal cell wall components. Such molecular patterns can also occurin other molecules such as plant alkaloids. These targets of innateimmune recognition are called Pathogen Associated Molecular Patterns(PAMPs) since they are produced by microorganisms and not by theinfected host organism (Janeway et al., 1989; Medzhitov et al., 1997).

The receptors of the innate immune system that recognize PAMPs arecalled Pattern Recognition Receptors (PRRs) (Janeway et al., 1989;Medzhitov et al., 1997). These receptors vary in structure and belong toseveral different protein families. Some of these receptors recognizePAMPs directly (e.g., CD14, DEC205, collectins), while others (e.g.,complement receptors) recognize the products generated by PAMPrecognition. Members of these receptor families can, generally, bedivided into three types: 1) humoral receptors circulating in theplasma; 2) endocytic receptors expressed on immune-cell surfaces, and 3)signaling receptors that can be expressed either on the cell surface orintracellularly (Medzhitov et al., 1997; Fearon et al., 1996).

Cellular PRRs are expressed on effector cells of the innate immunesystem, including cells that function as professional antigen-presentingcells (APC) in adaptive immunity. Such effector cells include, but arenot limited to, macrophages, dendritic cells, B lymphocytes and surfaceepithelia. This expression profile allows PRRs to directly induce innateeffector mechanisms, and also to alert the host organism to the presenceof infectious agents by inducing the expression of a set of endogenoussignals, such as inflammatory cytokines and chemokines, as discussedbelow. This latter function allows efficient mobilization of effectorforces to combat the invaders.

The primary function of dendritic cells (DCs) is to acquire antigen inthe peripheral tissues, travel to secondary lymphoid tissue, and presentantigen to effector T cells of the immune system (Banchereau, et al.,2000; Banchereau, et al., 1998). As DCs carry out their crucial role inthe immune response, they undergo maturational changes allowing them toperform the appropriate function for each environment (Termeer, C. C. etal., 2000). During DC maturation, antigen uptake potential is lost, thesurface density of major histocompatibility complex (MHC) class I andclass II molecules increases by 10-100 fold, and CD40, costimulatory andadhesion molecule expression also greatly increases (Lanzavecchia, A. etal., 2000). In addition, other genetic alterations permit the DCs tohome to the T cell-rich paracortex of draining lymph nodes and toexpress T-cell chemokines that attract naïve and memory T cells andprime antigen-specific naïve TH0 cells (Adema, G. J. et al., 1997).During this stage, mature DCs present antigen via their MHC II moleculesto CD4+ T helper cells, inducing the upregulation of T cell CD40 ligand(CD40L) that, in turn, engages the DC CD40 receptor. This DC:T cellinteraction induces rapid expression of additional DC molecules that arecrucial for the initiation of a potent CD8+ cytotoxic T lymphocyte (CTL)response, including further upregulation of MHC I and II molecules,adhesion molecules, costimulatory molecules (e.g., B7.1, B7.2),cytokines (e.g., IL-12) and anti-apoptotic proteins (e.g., Bcl-2)(Anderson, D. M., et al., 1997; Caux, C., et al., 1997; Ohshima, Y., etal., 1997; Sallusto, F., et al., 1998). CD8+ T cells exit lymph nodes,reenter circulation and home to the original site of inflammation todestroy pathogens or malignant cells.

One key parameter influencing the function of DCs is the CD40 receptor,serving as the “on switch” for DCs (Bennett, S. R. et al., 1998; Clark,S. R. et al., 2000; Fernandez, N.C., et al., 1999; Ridge, J. P. et al.,1998; Schoenberger, S. P., et al., 1998). CD40 is a 48-kDa transmembranemember of the TNF receptor superfamily (McWhirter, S. M., et al., 1999).CD40-CD40L interaction induces CD40 trimerization, necessary forinitiating signaling cascades involving TNF receptor associated factors(TRAFs) (Ni, C. Z., et al., 2000; Pullen, S. S. et al., 1999). CD40 usesthese signaling molecules to activate several transcription factors inDCs, including NF-kappa B, AP-1, STAT3, and p38MAPK (McWhirter, S. M.,et al., 1999).

The present invention contemplates a novel DC activation system based onrecruiting signaling molecules or co-stimulatory polypeptides to theplasmid membrane of the DCs resulting in prolonged/increased activationand/or survival in the DCs. Co-stimulatory polypeptides include anymolecule or polypeptide that activates the NFkappaB pathway, Aktpathway, and/or p38 pathway. The DC activation system is based uponutilizing a recombinant signaling molecule fused to a ligand-bindingdomains (i.e., a small molecule binding domain) in which theco-stimulatory polypeptide is activated and/or regulated with a ligandresulting in oligomerization (i.e., a lipid-permeable, organic,dimerizing drug). Other systems that may be used to crosslink oroligomerization of co-stimulatory polypeptides include antibodies,natural ligands, and/or artificial cross-reacting or synthetic ligands.Yet further, other dimerization systems contemplated include thecoumermycin/DNA gyrase B system.

Co-stimulatory polypeptides that can be used in the present inventioninclude those that activate NFkappaB and other variable signalingcascades for example the p38 pathway and/or Akt pathway. Suchco-stimulatory polypeptides include, but are not limited to PatternRecognition Receptors, C-reactive protein receptors (i.e., Nod1, Nod2,PtX3-R), TNF receptors (i.e., CD40, RANK/TRANCE-R, OX40, 4-1BB), and HSPreceptors (Lox-1 and CD-91). Pattern Recognition Receptors include, butare not limited to endocytic pattern-recognition receptors (i.e.,mannose receptors, scavenger receptors (i.e., Mac-1, LRP, peptidoglycan,techoic acids, toxins, CD11c/CR4)); external signal pattern-recognitionreceptors (Toll-like receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,TLR7, TLR8, TLR9, TLR10), peptidoglycan recognition protein, (PGRPs bindbacterial peptidoglycan, and CD14); internal signal pattern-recognitionreceptors (i.e., NOD-receptors 1 & 2), RIG1, and PRRs shown in FIG. 28 .Those of ordinary skill in the art are also aware of other PatternRecognition Receptors suitable for the present invention, includingthose discussed in, for example, Werts C., et al., Cell Death andDifferentiation (2006) 13:798-815; Meylan, E., et al., Nature (2006)442:39-44; and Strober, W., et al., Nature Reviews (2006) 6:9-20.

III. Engineering Expression Constructs

The present invention involves an expression construct encoding aco-stimulatory polypeptide and a ligand-binding domain, all operativelylinked. More particularly, more than one ligand-binding domain is usedin the expression construct. Yet further, the expression constructcontains a membrane-targeting sequence. One with skill in the artrealizes that appropriate expression constructs may include aco-stimulatory polypeptide element on either side of the above FKBPligand-binding elements. The expression construct of the presentinvention may be inserted into a vector, for example a viral vector orplasmid.

A. Co-stimulatory Polypeptides

In the present invention, co-stimulatory polypeptide molecules arecapable of amplifying the T-cell-mediated response by upregulatingdendritic cell expression of antigen presentation molecules.Co-stimulatory proteins that are contemplated in the present inventioninclude, for example, but are not limited to the members of tumornecrosis factor (TNF) family (i.e., CD40, RANK/TRANCE-R, OX40, 4-1B),Toll-like receptors, C-reactive protein receptors, Pattern RecognitionReceptors, and HSP receptors. Typically, the cytoplasmic domains fromthese co-stimulatory polypeptides are used in the expression vector. Thecytoplasmic domain from one of the various co-stimulatory polypeptides,including mutants thereof, where the recognition sequence involved ininitiating transcription associated with the cytoplasmic domain is knownor a gene responsive to such sequence is known.

In specific embodiments of the present invention, the co-stimulatorypolypeptide molecule is CD40. The CD40 molecule comprises a nucleic acidmolecule which: (1) hybridizes under stringent conditions to a nucleicacid having the sequence of a known CD40 gene and (2) codes for a CD40polypeptide. Preferably the CD40 polypeptide is lacking theextracellular domain. Exemplary polynucleotide sequences that encodeCD40 polypeptides include, but are not limited to SEQ.ID.NO: 1 and CD40isoforms from other species. It is contemplated that other normal ormutant variants of CD40 can be used in the present invention. Thus, aCD40 region can have an amino acid sequence that differs from the nativesequence by one or more amino acid substitutions, deletions and/orinsertions. For example, one or more TNF receptor associated factor(TRAF) binding regions may be eliminated or effectively eliminated(e.g., a CD40 amino acid sequence is deleted or altered such that a TRAFprotein does not bind or binds with lower affinity than it binds to thenative CD40 sequence). In particular embodiments, a TRAF 3 bindingregion is deleted or altered such that it is eliminated or effectivelyeliminated (e.g., amino acids 250-254 may be altered or deleted; Haueret al., PNAS 102(8): 2874-2879 (2005)).

In certain embodiments, the present invention involves the manipulationof genetic material to produce expression constructs that encode aninducible form of CD40 (iCD40). Such methods involve the generation ofexpression constructs containing, for example, a heterologous nucleicacid sequence encoding CD40 cytoplasmic domain and a means for itsexpression, replicating the vector in an appropriate helper cell,obtaining viral particles produced therefrom, and infecting cells withthe recombinant virus particles.

Thus, the preferable CD40 molecule of the present invention lacks theextracellular domain. In specific embodiments, the extracellular domainis truncated or removed. It is also contemplated that the extracellulardomain can be mutated using standard mutagenesis, insertions, deletions,or substitutions to produce an CD40 molecule that does not have afunctional extracellular domain. A CD40 nucleic acid may have thenucleic acid sequence of SEQ.ID.NO: 1. The CD40 nucleic acids of theinvention also include homologs and alleles of a nucleic acid having thesequence of SEQ.ID.NO: 1, as well as, functionally equivalent fragments,variants, and analogs of the foregoing nucleic acids.

In the context of gene therapy, the gene will be a heterologouspolynucleotide sequence derived from a source other than the viralgenome, which provides the backbone of the vector. The gene is derivedfrom a prokaryotic or eukaryotic source such as a bacterium, a virus,yeast, a parasite, a plant, or even an animal. The heterologous DNA alsois derived from more than one source, i.e., a multigene construct or afusion protein. The heterologous DNA also may include a regulatorysequence, which is derived from one source and the gene from a differentsource.

B. Ligand-binding Regions

The ligand-binding (“dimerization”) domain of the expression constructof this invention can be any convenient domain that will allow forinduction using a natural or unnatural ligand, preferably an unnaturalsynthetic ligand. The ligand-binding domain can be internal or externalto the cellular membrane, depending upon the nature of the construct andthe choice of ligand. A wide variety of ligand-binding proteins,including receptors, are known, including ligand-binding proteinsassociated with the cytoplasmic regions indicated above. As used hereinthe term “ligand-binding domain can be interchangeable with the term“receptor”. Of particular interest are ligand-binding proteins for whichligands (preferably small organic ligands) are known or may be readilyproduced. These ligand-binding domains or receptors include the FKBPsand cyclophilin receptors, the steroid receptors, the tetracyclinereceptor, the other receptors indicated above, and the like, as well as“unnatural” receptors, which can be obtained from antibodies,particularly the heavy or light chain subunit, mutated sequencesthereof, random amino acid sequences obtained by stochastic procedures,combinatorial syntheses, and the like.

For the most part, the ligand-binding domains or receptor domains willbe at least about amino acids, and fewer than about 350 amino acids,usually fewer than 200 amino acids, either as the natural domain ortruncated active portion thereof. Preferably the binding domain will besmall (<25 kDa, to allow efficient transfection in viral vectors),monomeric (this rules out the avidin-biotin system), nonimmunogenic, andshould have synthetically accessible, cell permeable, nontoxic ligandsthat can be configured for dimerization.

The receptor domain can be intracellular or extracellular depending uponthe design of the expression construct and the availability of anappropriate ligand. For hydrophobic ligands, the binding domain can beon either side of the membrane, but for hydrophilic ligands,particularly protein ligands, the binding domain will usually beexternal to the cell membrane, unless there is a transport system forinternalizing the ligand in a form in which it is available for binding.For an intracellular receptor, the construct can encode a signal peptideand transmembrane domain 5′ or 3′ of the receptor domain sequence or byhaving a lipid attachment signal sequence 5′ of the receptor domainsequence. Where the receptor domain is between the signal peptide andthe transmembrane domain, the receptor domain will be extracellular.

The portion of the expression construct encoding the receptor can besubjected to mutagenesis for a variety of reasons. The mutagenizedprotein can provide for higher binding affinity, allow fordiscrimination by the ligand of the naturally occurring receptor and themutagenized receptor, provide opportunities to design a receptor-ligandpair, or the like. The change in the receptor can involve changes inamino acids known to be at the binding site, random mutagenesis usingcombinatorial techniques, where the codons for the amino acidsassociated with the binding site or other amino acids associated withconformational changes can be subject to mutagenesis by changing thecodon(s) for the particular amino acid, either with known changes orrandomly, expressing the resulting proteins in an appropriateprokaryotic host and then screening the resulting proteins for binding

Antibodies and antibody subunits, e.g., heavy or light chain,particularly fragments, more particularly all or part of the variableregion, or fusions of heavy and light chain to create high-affinitybinding, can be used as the binding domain. Antibodies that arecontemplated in the present invention include ones that are anectopically expressed human product, such as an extracellular domainthat would not trigger an immune response and generally not expressed inthe periphery (i.e., outside the CNS/brain area). Such examples,include, but are not limited to low affinity nerve growth factorreceptor (LNGFR), and embryonic surface proteins (i.e., carcinoembryonicantigen).

Yet further, antibodies can be prepared against haptenic molecules,which are physiologically acceptable, and the individual antibodysubunits screened for binding affinity. The cDNA encoding the subunitscan be isolated and modified by deletion of the constant region,portions of the variable region, mutagenesis of the variable region, orthe like, to obtain a binding protein domain that has the appropriateaffinity for the ligand. In this way, almost any physiologicallyacceptable haptenic compound can be employed as the ligand or to providean epitope for the ligand. Instead of antibody units, natural receptorscan be employed, where the binding domain is known and there is a usefulligand for binding.

C. Oligomerization

The transduced signal will normally result from ligand-mediatedoligomerization of the chimeric protein molecules, i.e., as a result ofoligomerization following ligand-binding, although other binding events,for example allosteric activation, can be employed to initiate a signal.The construct of the chimeric protein will vary as to the order of thevarious domains and the number of repeats of an individual domain.

For multimerizing the receptor, the ligand for the ligand-bindingdomains/receptor domains of the chimeric surface membrane proteins willusually be multimeric in the sense that it will have at least twobinding sites, with each of the binding sites capable of binding to thereceptor domain. Desirably, the subject ligands will be a dimer orhigher order oligomer, usually not greater than about tetrameric, ofsmall synthetic organic molecules, the individual molecules typicallybeing at least about 150 D and fewer than about 5 kDa, usually fewerthan about 3 kDa. A variety of pairs of synthetic ligands and receptorscan be employed. For example, in embodiments involving naturalreceptors, dimeric FK506 can be used with an FKBP receptor, dimerizedcyclosporin A can be used with the cyclophilin receptor, dimerizedestrogen with an estrogen receptor, dimerized glucocorticoids with aglucocorticoid receptor, dimerized tetracycline with the tetracyclinereceptor, dimerized vitamin D with the vitamin D receptor, and the like.Alternatively higher orders of the ligands, e.g., trimeric can be used.For embodiments involving unnatural receptors, e.g., antibody subunits,modified antibody subunits or modified receptors and the like, any of alarge variety of compounds can be used. A significant characteristic ofthese ligand units is that they bind the receptor with high affinity andare able to be dimerized chemically.

In certain embodiments, the present invention utilizes the technique ofchemically induced dimerization (CID) to produce a conditionallycontrolled protein or polypeptide. In addition to this technique beinginducible, it also is reversible, due to the degradation of the labiledimerizing agent or administration of a monomeric competitive inhibitor.

CID system uses synthetic bivalent ligands to rapidly crosslinksignaling molecules that are fused to ligand-binding domains CID. Thissystem has been used to trigger the oligomerization and activation ofcell surface (Spencer et al., 1993; Spencer et al., 1996; Blau et al.,1997), or cytosolic proteins (Luo et al., 1996; MacCorkle et al., 1998),the recruitment of transcription factors to DNA elements to modulatetranscription (Ho et al., 1996; Rivera et al., 1996) or the recruitmentof signaling molecules to the plasma membrane to stimulate signaling(Spencer et al., 1995; Holsinger et al., 1995).

The CID system is based upon the notion that surface receptoraggregation effectively activates downstream signaling cascades. In thesimplest embodiment, the CID system uses a dimeric analog of the lipidpermeable immunosuppressant drug, FK506, which loses its normalbioactivity while gaining the ability to crosslink molecules geneticallyfused to the FK506-binding protein, FKBP12. By fusing one or more FKBPsand a myristoylation sequence to the cytoplasmic signaling domain of atarget receptor, one can stimulate signaling in a dimerizerdrug-dependent, but ligand and ectodomain-independent manner. Thisprovides the system with temporal control, reversibility using monomericdrug analogs, and enhanced specificity. The high affinity ofthird-generation AP20187/AP1903 CIDs for their binding domain, FKBP12permits specific activation of the recombinant receptor in vivo withoutthe induction of non-specific side effects through endogenous FKBP12. Inaddition, the synthetic ligands are resistant to protease degradation,making them more efficient at activating receptors in vivo than mostdelivered protein agents.

The ligands used in the present invention are capable of binding to twoor more of the ligand-binding domains. One skilled in the art realizesthat the chimeric proteins may be able to bind to more than one ligandwhen they contain more than one ligand-binding domain. The ligand istypically a non-protein or a chemical. Exemplary ligands include, butare not limited to dimeric FK506 (e.g., FK1012).

Since the mechanism of CD40 activation is fundamentally based ontrimerization, this receptor is particularly amenable to the CID system.CID regulation provides the system with 1) temporal control, 2)reversibility by addition of a non-active monomer upon signs of anautoimmune reaction, and 3) limited potential for non-specific sideeffects. In addition, inducible in vivo DC CD40 activation wouldcircumvent the requirement of a second “danger” signal normally requiredfor complete induction of CD40 signaling and would potentially promoteDC survival in situ allowing for enhanced T cell priming. Thus,engineering DC vaccines to express iCD40 amplifies the T cell-mediatedkilling response by upregulating DC expression of antigen presentationmolecules, adhesion molecules, TH1 promoting cytokines, and pro-survivalfactors.

Other dimerization systems contemplated include the coumermycin/DNAgyrase B system. Coumermycin-induced dimerization activates a modifiedRaf protein and stimulates the MAP kinase cascade. See Farrar et al.,1996.

D. Membrane-Targeting

A membrane-targeting sequence provides for transport of the chimericprotein to the cell surface membrane, where the same or other sequencescan encode binding of the chimeric protein to the cell surface membrane.Molecules in association with cell membranes contain certain regionsthat facilitate the membrane association, and such regions can beincorporated into a chimeric protein molecule to generatemembrane-targeted molecules. For example, some proteins containsequences at the N-terminus or C-terminus that are acylated, and theseacyl moieties facilitate membrane association. Such sequences arerecognized by acyltransferases and often conform to a particularsequence motif. Certain acylation motifs are capable of being modifiedwith a single acyl moiety and others are capable of being modified withmultiple acyl moieties. For example the N-terminal sequence of theprotein kinase Src can comprise a single myristoyl moiety. Dualacylation regions are located within the N-terminal regions of certainprotein kinases (e.g., Yes, Fyn, Lck) and G-protein alpha subunits. Suchdual acylation regions often are located within the first eighteen aminoacids of such proteins, and conform to the sequence motifMet-Gly-Cys-Xaa-Cys (SEQ ID NO: 7), where the Met is cleaved, the Gly isN-acylated and one of the Cys residues is S-acylated. The Gly often ismyristoylated and a Cys can be palmitoylated. Acylation regionsconforming to the sequence motif Cys-Ala-Ala-Xaa (so called “CAAXboxes”), which can be modified with C15 or C10 isoprenyl moieties, fromthe C-terminus of G-protein gamma subunits and other proteins (e.g.,World Wide Web address ebi.ac.uk/interpro/DisplayIproEntry?ac=IPR001230)also can be utilized. These and other acylation motifs are known to theperson of ordinary skill in the art (e.g., Gauthier-Campbell et al.,Molecular Biology of the Cell 15: 2205-2217 (2004); Glabati et al.,Biochem. J. 303: 697-700 (1994) and Zlakine et al., J. Cell Science 110:673-679 (1997)), and can be incorporated in chimeric molecules to inducemembrane localization. In certain embodiments, a native sequence from aprotein containing an acylation motif is incorporated into a chimericprotein. For example, in some embodiments, an N-terminal portion of Lck,Fyn or Yes or a G-protein alpha subunit, such as the first twenty-fiveN-terminal amino acids or fewer from such proteins (e.g., about 5 toabout 20 amino acids, about 10 to about 19 amino acids, or about 15 toabout 19 amino acids of the native sequence with optional mutations),may be incorporated within the N-terminus of a chimeric protein. Incertain embodiments, a C-terminal sequence of about 25 amino acids orless from a G-protein gamma subunit containing a CAAX box motif sequence(e.g., about 5 to about 20 amino acids, about 10 to about 18 aminoacids, or about 15 to about 18 amino acids of the native sequence withoptional mutations) can be linked to the C-terminus of a chimericprotein.

In some embodiments, an acyl moiety has a log p value of +1 to +6, andsometimes has a log p value of +3 to +4.5. Log p values are a measure ofhydrophobicity and often are derived from octanol/water partitioningstudies, in which molecules with higher hydrophobicity partition intooctanol with higher frequency and are characterized as having a higherlog p value. Log p values are published for a number of lipophilicmolecules and log p values can be calculated using known partitioningprocesses (e.g., Chemical Reviews, Vol. 71, Issue 6, page 599, whereentry 4493 shows lauric acid having a log p value of 4.2). Any acylmoiety can be linked to a peptide composition described above and testedfor antimicrobial activity using known methods and those describedhereafter. The acyl moiety sometimes is a C1-C20 alkyl, C2-C20 alkenyl,C2-C20 alkynyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C4-C12cyclalkylalkyl, aryl, substituted aryl, or aryl (C1-C₄) alkyl, forexample. Any acyl-containing moiety sometimes is a fatty acid, andexamples of fatty acid moieties are propyl (C3), butyl (C4), pentyl(C5), hexyl (C6), heptyl (C7), octyl (C8), nonyl (C9), decyl (C10),undecyl (C11), lauryl (C12), myristyl (C14), palmityl (C16), stearyl(C18), arachidyl (C20), behenyl (C22) and lignoceryl moieties (C24), andeach moiety can contain 0, 1, 2, 3, 4, 5, 6, 7 or 8 unsaturations (i.e.,double bonds). An acyl moiety sometimes is a lipid molecule, such as aphosphatidyl lipid (e.g., phosphatidyl serine, phosphatidyl inositol,phosphatidyl ethanolamine, phosphatidyl choline), sphingolipid (e.g.,shingomyelin, sphigosine, ceramide, ganglioside, cerebroside), ormodified versions thereof. In certain embodiments, one, two, three, fouror five or more acyl moieties are linked to a membrane associationregion.

A chimeric protein herein also may include a single-pass or multiplepass transmembrane sequence (e.g., at the N-terminus or C-terminus ofthe chimeric protein). Single pass transmembrane regions are found incertain CD molecules, tyrosine kinase receptors, serine/threonine kinasereceptors, TGFbeta, BMP, activin and phosphatases. Single passtransmembrane regions often include a signal peptide region and atransmembrane region of about 20 to about 25 amino acids, many of whichare hydrophobic amino acids and can form an alpha helix. A short trackof positively charged amino acids often follows the transmembrane span.Multiple pass proteins include ion pumps, ion channels, andtransporters, and include two or more helices that span the membranemultiple times. All or substantially all of a multiple pass proteinsometimes is incorporated in a chimeric protein. Sequences for singlepass and multiple pass transmembrane regions are known and can beselected for incorporation into a chimeric protein molecule by theperson of ordinary skill in the art.

Any membrane-targeting sequence can be employed that is functional inthe host and may, or may not, be associated with one of the otherdomains of the chimeric protein. In some embodiments of the invention,such sequences include, but are not limited to myristoylation-targetingsequence, palmitoylation targeting sequence, prenylation sequences(i.e., farnesylation, geranyl-geranylation, CAAX Box) or transmembranesequences (utilizing signal peptides) from receptors.

E. Selectable Markers

In certain embodiments of the invention, the expression constructs ofthe present invention contain nucleic acid constructs whose expressionis identified in vitro or in vivo by including a marker in theexpression construct. Such markers would confer an identifiable changeto the cell permitting easy identification of cells containing theexpression construct. Usually the inclusion of a drug selection markeraids in cloning and in the selection of transformants. For example,genes that confer resistance to neomycin, puromycin, hygromycin, DHFR,GPT, zeocin and histidinol are useful selectable markers. Alternatively,enzymes such as herpes simplex virus thymidine kinase (tk) are employed.Immunologic markers also can be employed. The selectable marker employedis not believed to be important, so long as it is capable of beingexpressed simultaneously with the nucleic acid encoding a gene product.Further examples of selectable markers are well known to one of skill inthe art and include reporters such as EGFP, beta-gal or chloramphenicolacetyltransferase (CAT).

F. Control Regions

-   -   1. Promoters

The particular promoter employed to control the expression of apolynucleotide sequence of interest is not believed to be important, solong as it is capable of directing the expression of the polynucleotidein the targeted cell. Thus, where a human cell is targeted, it ispreferable to position the polynucleotide sequence-coding regionadjacent to and under the control of a promoter that is capable of beingexpressed in a human cell. Generally speaking, such a promoter mightinclude either a human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, β-actin, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it is desirable to prohibit orreduce expression of one or more of the transgenes. Examples oftransgenes that are toxic to the producer cell line are pro-apoptoticand cytokine genes. Several inducible promoter systems are available forproduction of viral vectors where the transgene products are toxic (addin more inducible promoters).

The ecdysone system (Invitrogen, Carlsbad, CA) is one such system. Thissystem is designed to allow regulated expression of a gene of interestin mammalian cells. It consists of a tightly regulated expressionmechanism that allows virtually no basal level expression of thetransgene, but over 200-fold inducibility. The system is based on theheterodimeric ecdysone receptor of Drosophila, and when ecdysone or ananalog such as muristerone A binds to the receptor, the receptoractivates a promoter to turn on expression of the downstream transgenehigh levels of mRNA transcripts are attained. In this system, bothmonomers of the heterodimeric receptor are constitutively expressed fromone vector, whereas the ecdysone-responsive promoter, which drivesexpression of the gene of interest is on another plasmid. Engineering ofthis type of system into the gene transfer vector of interest wouldtherefore be useful. Cotransfection of plasmids containing the gene ofinterest and the receptor monomers in the producer cell line would thenallow for the production of the gene transfer vector without expressionof a potentially toxic transgene. At the appropriate time, expression ofthe transgene could be activated with ecdysone or muristeron A.

Another inducible system that would be useful is the Tet-Off™ or Tet-On™system (Clontech, Palo Alto, CA) originally developed by Gossen andBujard (Gossen and Bujard, 1992; Gossen et al., 1995). This system alsoallows high levels of gene expression to be regulated in response totetracycline or tetracycline derivatives such as doxycycline. In theTet-On™ system, gene expression is turned on in the presence ofdoxycycline, whereas in the Tet-Off™ system, gene expression is turnedon in the absence of doxycycline. These systems are based on tworegulatory elements derived from the tetracycline resistance operon ofE. coli. The tetracycline operator sequence to which the tetracyclinerepressor binds, and the tetracycline repressor protein. The gene ofinterest is cloned into a plasmid behind a promoter that hastetracycline-responsive elements present in it. A second plasmidcontains a regulatory element called the tetracycline-controlledtransactivator, which is composed, in the Tet-Off™ system, of the VP16domain from the herpes simplex virus and the wild-type tertracyclinerepressor. Thus in the absence of doxycycline, transcription isconstitutively on. In the Tet-On™ system, the tetracycline repressor isnot wild type and in the presence of doxycycline activatestranscription. For gene therapy vector production, the Tet-Off™ systemwould be preferable so that the producer cells could be grown in thepresence of tetracycline or doxycycline and prevent expression of apotentially toxic transgene, but when the vector is introduced to thepatient, the gene expression would be constitutively on.

In some circumstances, it is desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity are utilized depending onthe level of expression desired. In mammalian cells, the CMV immediateearly promoter if often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoietic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that are used depending on the desiredeffect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoterssuch as from the ElA, E₂A, or MLP region, AAV LTR, HSV-TK, and aviansarcoma virus.

Similarly tissue specific promoters are used to effect transcription inspecific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA associated promoter or prostate-specific glandularkallikrein.

In certain indications, it is desirable to activate transcription atspecific times after administration of the gene therapy vector. This isdone with such promoters as those that are hormone or cytokineregulatable. Cytokine and inflammatory protein responsive promoters thatcan be used include K and T kininogen (Kageyama et al., 1987), c-fos,TNF-alpha, C-reactive protein (Arcone et al., 1988), haptoglobin(Oliviero et al., 1987), serum amyloid A2, C/EBP alpha, IL-1, IL-6 (Poliand Cortese, 1989), Complement C3 (Wilson et al., 1990), IL-8, alpha-1acid glycoprotein (Prowse and Baumann, 1988), alpha-1 antitrypsin,lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et al.,1991), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UVradiation, retinoic acid, and hydrogen peroxide), collagenase (inducedby phorbol esters and retinoic acid), metallothionein (heavy metal andglucocorticoid inducible), Stromelysin (inducible by phorbol ester,interleukin-1 and EGF), alpha-2 macroglobulin and alpha-1anti-chymotrypsin.

It is envisioned that any of the above promoters alone or in combinationwith another can be useful according to the present invention dependingon the action desired. In addition, this list of promoters should not beconstrued to be exhaustive or limiting, those of skill in the art willknow of other promoters that are used in conjunction with the promotersand methods disclosed herein.

-   -   2. Enhancers

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNAEnhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization.

Any promoter/enhancer combination (as per the Eukaryotic Promoter DataBase EPDB) can be used to drive expression of the gene. Eukaryotic cellscan support cytoplasmic transcription from certain bacterial promotersif the appropriate bacterial polymerase is provided, either as part ofthe delivery complex or as an additional genetic expression construct.

-   -   3. Polyadenylation Signals

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence is employed such as human or bovine growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

-   -   4. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be in-frame with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements is used to create multigene, orpolycistronic messages. IRES elements are able to bypass theribosome-scanning model of 5′ methylated cap-dependent translation andbegin translation at internal sites (Pelletier and Sonenberg, 1988).IRES elements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

IV. Methods of Gene Transfer

In order to mediate the effect of the transgene expression in a cell, itwill be necessary to transfer the expression constructs of the presentinvention into a cell. Such transfer may employ viral or non-viralmethods of gene transfer. This section provides a discussion of methodsand compositions of gene transfer.

A transformed cell comprising an expression vector is generated byintroducing into the cell the expression vector. Suitable methods forpolynucleotide delivery for transformation of an organelle, a cell, atissue or an organism for use with the current invention includevirtually any method by which a polynucleotide (e.g., DNA) can beintroduced into an organelle, a cell, a tissue or an organism, asdescribed herein or as would be known to one of ordinary skill in theart.

A host cell can, and has been, used as a recipient for vectors. Hostcells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded polynucleotide sequences. Numerouscell lines and cultures are available for use as a host cell, and theycan be obtained through the American Type Culture Collection (ATCC),which is an organization that serves as an archive for living culturesand genetic materials. In specific embodiments, the host cell is adendritic cell, which is an antigen-presenting cell.

It is well within the knowledge and skill of a skilled artisan todetermine an appropriate host. Generally this is based on the vectorbackbone and the desired result. A plasmid or cosmid, for example, canbe introduced into a prokaryote host cell for replication of manyvectors. Bacterial cells used as host cells for vector replicationand/or expression include DH5alpha, JM109, and KCB, as well as a numberof commercially available bacterial hosts such as SURE® Competent Cellsand SOLOPACK Gold Cells (STRATAGENE®, La Jolla, CA). Alternatively,bacterial cells such as E. coli LE392 could be used as host cells forphage viruses. Eukaryotic cells that can be used as host cells include,but are not limited to yeast, insects and mammals. Examples of mammalianeukaryotic host cells for replication and/or expression of a vectorinclude, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO,Saos, and PC12. Examples of yeast strains include, but are not limitedto, YPH499, YPH500 and YPH501.

A. Non-viral Transfer

-   -   1. Ex Vivo Transformation

Methods for transfecting vascular cells and tissues removed from anorganism in an ex vivo setting are known to those of skill in the art.For example, canine endothelial cells have been genetically altered byretroviral gene transfer in vitro and transplanted into a canine (Wilsonet al., 1989). In another example, Yucatan minipig endothelial cellswere transfected by retrovirus in vitro and transplanted into an arteryusing a double-balloon catheter (Nabel et al., 1989). Thus, it iscontemplated that cells or tissues may be removed and transfected exvivo using the polynucleotides of the present invention. In particularaspects, the transplanted cells or tissues may be placed into anorganism. Thus, it is well within the knowledge of one skilled in theart to isolate dendritic cells from an animal, transfect the cells withthe expression vector and then administer the transfected or transformedcells back to the animal.

-   -   2. Injection

In certain embodiments, a polynucleotide may be delivered to anorganelle, a cell, a tissue or an organism via one or more injections(i.e., a needle injection), such as, for example, subcutaneously,intradermally, intramuscularly, intravenously, intraperitoneally, etc.Methods of injection of vaccines are well known to those of ordinaryskill in the art (e.g., injection of a composition comprising a salinesolution). Further embodiments of the present invention include theintroduction of a polynucleotide by direct microinjection. The amount ofthe expression vector used may vary upon the nature of the antigen aswell as the organelle, cell, tissue or organism used.

Intradermal, intranodal, or intralymphatic injections are some of themore commonly used methods of DC administration. Intradermal injectionis characterized by a low rate of absorption into the bloodstream butrapid uptake into the lymphatic system. The presence of large numbers ofLangerhans dendritic cells in the dermis will transport intact as wellas processed antigen to draining lymph nodes. Proper site preparation isnecessary to perform this correctly (i.e., hair must be clipped in orderto observe proper needle placement). Intranodal injection allows fordirect delivery of antigen to lymphoid tissues. Intralymphatic injectionallows direct administration of DCs.

-   -   3. Electroporation

In certain embodiments of the present invention, a polynucleotide isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high-voltage electric discharge. In some variantsof this method, certain cell wall-degrading enzymes, such aspectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells (U.S. Pat. No. 5,384,253, incorporated herein byreference).

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

-   -   4. Calcium Phosphate

In other embodiments of the present invention, a polynucleotide isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

-   -   5. DEAE-Dextran

In another embodiment, a polynucleotide is delivered into a cell usingDEAE-dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, 1985).

-   -   6. Sonication Loading

Additional embodiments of the present invention include the introductionof a polynucleotide by direct sonic loading. LTK-fibroblasts have beentransfected with the thymidine kinase gene by sonication loading(Fechheimer et al., 1987).

-   -   7. Liposome-Mediated Transfection

In a further embodiment of the invention, a polynucleotide may beentrapped in a lipid complex such as, for example, a liposome. Liposomesare vesicular structures characterized by a phospholipid bilayermembrane and an inner aqueous medium. Multilamellar liposomes havemultiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers (Ghosh and Bachhawat, 1991). Alsocontemplated is a polynucleotide complexed with Lipofectamine (GibcoBRL) or Superfect (Qiagen).

-   -   8. Receptor Mediated Transfection

Still further, a polynucleotide may be delivered to a target cell viareceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis thatwill be occurring in a target cell. In view of the cell type-specificdistribution of various receptors, this delivery method adds anotherdegree of specificity to the present invention.

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a polynucleotide-binding agent. Otherscomprise a cell receptor-specific ligand to which the polynucleotide tobe delivered has been operatively attached. Several ligands have beenused for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner etal., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishesthe operability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand is chosen to correspond to a receptor specificallyexpressed on the target cell population.

In other embodiments, a polynucleotide delivery vehicle component of acell-specific polynucleotide-targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The polynucleotide(s) tobe delivered are housed within the liposome and the specific bindingligand is functionally incorporated into the liposome membrane. Theliposome will thus specifically bind to the receptor(s) of a target celland deliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor-mediated delivery of a polynucleotide tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the polynucleotide delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialoganglioside, have been incorporated intoliposomes and observed an increase in the uptake of the insulin gene byhepatocytes (Nicolau et al., 1987). It is contemplated that thetissue-specific transforming constructs of the present invention can bespecifically delivered into a target cell in a similar manner.

-   -   9. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce apolynucleotide into at least one, organelle, cell, tissue or organism(U.S. Pat. Nos. 5,550,318; 5,538,880; 5,610,042; and PCT Application WO94/09699; each of which is incorporated herein by reference). Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). There are a widevariety of microprojectile bombardment techniques known in the art, manyof which are applicable to the invention.

In this microprojectile bombardment, one or more particles may be coatedwith at least one polynucleotide and delivered into cells by apropelling force. Several devices for accelerating small particles havebeen developed. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold particles orbeads. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA-coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

B. Viral Vector-Mediated Transfer

In certain embodiments, transgene is incorporated into a viral particleto mediate gene transfer to a cell. Typically, the virus simply will beexposed to the appropriate host cell under physiologic conditions,permitting uptake of the virus. The present methods are advantageouslyemployed using a variety of viral vectors, as discussed below.

-   -   1. Adenovirus

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized DNA genome, ease of manipulation, high titer,wide target-cell range, and high infectivity. The roughly 36 kb viralgenome is bounded by 100-200 base pair (bp) inverted terminal repeats(ITR), in which are contained cis-acting elements necessary for viralDNA replication and packaging. The early (E) and late (L) regions of thegenome that contain different transcription units are divided by theonset of viral DNA replication.

The E1 region (E1A and E1B) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication. These proteins areinvolved in DNA replication, late gene expression, and host cell shutoff (Renan, 1990). The products of the late genes (L1, L2, L3, L4 andL5), including the majority of the viral capsid proteins, are expressedonly after significant processing of a single primary transcript issuedby the major late promoter (MLP). The MLP (located at 16.8 map units) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5

tripartite leader (TL) sequence, which makes them preferred mRNAs fortranslation.

In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.The two goals are, to an extent, coterminous in that elimination ofadenoviral genes serves both ends. By practice of the present invention,it is possible achieve both these goals while retaining the ability tomanipulate the therapeutic constructs with relative ease.

The large displacement of DNA is possible because the cis elementsrequired for viral DNA replication all are localized in the invertedterminal repeats (ITR) (100-200 bp) at either end of the linear viralgenome. Plasmids containing ITR's can replicate in the presence of anon-defective adenovirus (Hay et al., 1984). Therefore, inclusion ofthese elements in an adenoviral vector should permit replication.

In addition, the packaging signal for viral encapsulation is localizedbetween 194-385 bp (0.5-1.1 map units) at the left end of the viralgenome (Hearing et al., 1987). This signal mimics the proteinrecognition site in bacteriophage lambda DNA where a specific sequenceclose to the left end, but outside the cohesive end sequence, mediatesthe binding to proteins that are required for insertion of the DNA intothe head structure. E1 substitution vectors of Ad have demonstrated thata 450 bp (0-1.25 map units) fragment at the left end of the viral genomecould direct packaging in 293 cells (Levrero et al., 1991).

Previously, it has been shown that certain regions of the adenoviralgenome can be incorporated into the genome of mammalian cells and thegenes encoded thereby expressed. These cell lines are capable ofsupporting the replication of an adenoviral vector that is deficient inthe adenoviral function encoded by the cell line. There also have beenreports of complementation of replication deficient adenoviral vectorsby “helping” vectors, e.g., wild-type virus or conditionally defectivemutants.

Replication-deficient adenoviral vectors can be complemented, in trans,by helper virus. This observation alone does not permit isolation of thereplication-deficient vectors, however, since the presence of helpervirus, needed to provide replicative functions, would contaminate anypreparation. Thus, an additional element was needed that would addspecificity to the replication and/or packaging of thereplication-deficient vector. That element, as provided for in thepresent invention, derives from the packaging function of adenovirus.

It has been shown that a packaging signal for adenovirus exists in theleft end of the conventional adenovirus map (Tibbetts, 1977). Laterstudies showed that a mutant with a deletion in the E1A (194-358 bp)region of the genome grew poorly even in a cell line that complementedthe early (E1A) function (Hearing and Shenk, 1983). When a compensatingadenoviral DNA (0-353 bp) was recombined into the right end of themutant, the virus was packaged normally. Further mutational analysisidentified a short, repeated, position-dependent element in the left endof the Ad5 genome. One copy of the repeat was found to be sufficient forefficient packaging if present at either end of the genome, but not whenmoved towards the interior of the Ad5 DNA molecule (Hearing et al.,1987).

By using mutated versions of the packaging signal, it is possible tocreate helper viruses that are packaged with varying efficiencies.Typically, the mutations are point mutations or deletions. When helperviruses with low efficiency packaging are grown in helper cells, thevirus is packaged, albeit at reduced rates compared to wild-type virus,thereby permitting propagation of the helper. When these helper virusesare grown in cells along with virus that contains wild-type packagingsignals, however, the wild-type packaging signals are recognizedpreferentially over the mutated versions. Given a limiting amount ofpackaging factor, the virus containing the wild-type signals is packagedselectively when compared to the helpers. If the preference is greatenough, stocks approaching homogeneity should be achieved.

-   -   2. Retrovirus

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains threegenes—gag, pol and env—that code for capsid proteins, polymerase enzyme,and envelope components, respectively. A sequence found upstream fromthe gag gene, termed psi, functions as a signal for packaging of thegenome into virions. Two long terminal repeat (LTR) sequences arepresent at the 5′ and 3′ ends of the viral genome. These contain strongpromoter and enhancer sequences and also are required for integration inthe host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding apromoter is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol and envgenes but without the LTR and psi components is constructed (Mann etal., 1983). When a recombinant plasmid containing a human cDNA, togetherwith the retroviral LTR and psi sequences is introduced into this cellline (by calcium phosphate precipitation for example), the psi sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression of many types of retroviruses require the division of hostcells (Paskind et al., 1975).

An approach designed to allow specific targeting of retrovirus vectorsrecently was developed based on the chemical modification of aretrovirus by the chemical addition of galactose residues to the viralenvelope. This modification could permit the specific infection of cellssuch as hepatocytes via asialoglycoprotein receptors, should this bedesired.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, the infection of a variety of human cellsthat bore those surface antigens was demonstrated with an ecotropicvirus in vitro (Roux et al., 1989).

-   -   3. Adeno-Associated Virus

AAV utilizes a linear, single-stranded DNA of about 4700 base pairs.Inverted terminal repeats flank the genome. Two genes are present withinthe genome, giving rise to a number of distinct gene products. Thefirst, the cap gene, produces three different virion proteins (VP),designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes fournon-structural proteins (NS). One or more of these rep gene products isresponsible for transactivating AAV transcription.

The three promoters in AAV are designated by their location, in mapunits, in the genome. These are, from left to right, p5, p19 and p40.Transcription gives rise to six transcripts, two initiated at each ofthree promoters, with one of each pair being spliced. The splice site,derived from map units 42-46, is the same for each transcript. The fournon-structural proteins apparently are derived from the longer of thetranscripts, and three virion proteins all arise from the smallesttranscript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The bestcharacterized of the helpers is adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low-levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

The terminal repeats of the AAV vector can be obtained by restrictionendonuclease digestion of AAV or a plasmid such as p201, which containsa modified AAV genome (Samulski et al., 1987), or by other methods knownto the skilled artisan, including but not limited to chemical orenzymatic synthesis of the terminal repeats based upon the publishedsequence of AAV. The ordinarily skilled artisan can determine, bywell-known methods such as deletion analysis, the minimum sequence orpart of the AAV ITRs which is required to allow function, i.e., stableand site-specific integration. The ordinarily skilled artisan also candetermine which minor modifications of the sequence can be toleratedwhile maintaining the ability of the terminal repeats to direct stable,site-specific integration.

AAV-based vectors have proven to be safe and effective vehicles for genedelivery in vitro, and these vectors are being developed and tested inpre-clinical and clinical stages for a wide range of applications inpotential gene therapy, both ex vivo and in vivo (Carter and Flotte,1995; Chatterjee et al., 1995; Ferrari et al., 1996; Fisher et al.,1996; Flotte et al., 1993; Goodman et al., 1994; Kaplitt et al., 1994;1996, Kessler et al., 1996; Koeberl et al., 1997; Mizukami et al.,1996).

AAV-mediated efficient gene transfer and expression in the lung has ledto clinical trials for the treatment of cystic fibrosis (Carter andFlotte, 1995; Flotte et al., 1993). Similarly, the prospects fortreatment of muscular dystrophy by AAV-mediated gene delivery of thedystrophin gene to skeletal muscle, of Parkinson's disease by tyrosinehydroxylase gene delivery to the brain, of hemophilia B by Factor IXgene delivery to the liver, and potentially of myocardial infarction byvascular endothelial growth factor gene to the heart, appear promisingsince AAV-mediated transgene expression in these organs has recentlybeen shown to be highly efficient (Fisher et al., 1996; Flotte et al.,1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCown et al.,1996; Ping et al., 1996; Xiao et al., 1996).

-   -   4. Other Viral Vectors

Other viral vectors are employed as expression constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) canarypox virus, and herpes viruses are employed. These viruses offer severalfeatures for use in gene transfer into various mammalian cells.

Once the construct has been delivered into the cell, the nucleic acidencoding the transgene are positioned and expressed at different sites.In certain embodiments, the nucleic acid encoding the transgene isstably integrated into the genome of the cell. This integration is inthe cognate location and orientation via homologous recombination (genereplacement) or it is integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid isstably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

V Enhancement of an Immune Response

In certain embodiments, the present invention contemplates a novel DCactivation strategy that incorporates the manipulation of signalingco-stimulatory polypeptides that activate NF

B pathways, Akt pathways, and/or p38 pathways. This DC activation systemcan be used in conjunction with or without standard vaccines to enhancethe immune response since it replaces the requirement for CD4+ T cellhelp during APC activation (Bennett S. R. et al., 1998; Ridge, J. P. etal., 1998; Schoenberger, S. P., et al., 1998). Thus, the DC activationsystem of the present invention enhances immune responses bycircumventing the need for the generation of MHC class II-specificpeptides.

In specific embodiments, the DC activation is via CD40 activation. Thus,DC activation via endogenous CD40/CD40L interactions may be subject todownregulation due to negative feedback, leading rapidly to the “IL-12burn-out effect”. Within 7 to 10 hours after CD40 activation, analternatively spliced isoform of CD40 (type II) is produced as asecretable factor (Tone, M., et al., 2001). Type II CD40 may act as adominant negative receptor, downregulating signaling through CD40L andpotentially limiting the potency of the immune response generated.Therefore, the present invention co-opts the natural regulation of CD40by creating an inducible form of CD40 (iCD40), lacking the extracellulardomain and activated instead by synthetic dimerizing ligands (Spencer,D. M. et al., 1993) through a technology termed chemically induceddimerization (CID).

The present invention comprises a method of enhancing the immuneresponse in an subject comprising the step of administering either theexpression vector, expression construct or transduced antigen-presentingcells of the present invention to the subject. The expression vector ofthe present invention encodes a co-stimulatory polypeptide, such asiCD40.

In certain embodiments the antigen-presenting cells are comprised in ananimal, such as human, non-human primate, cow, horse, pig, sheep, goat,dog, cat, or rodent. The subject is a human, more preferably, a patientsuffering from an infectious disease, and/or a subject that isimmunocompromised, or is suffering from a hyperproliferative disease.

In further embodiments of the present invention, the expressionconstruct and/or expression vector can be utilized as a composition orsubstance that activates antigen-presenting cells. Such a compositionthat “activates antigen-presenting cells” or “enhances the activityantigen-presenting cells” refers to the ability to stimulate one or moreactivities associated with antigen-presenting cells. Such activities arewell known by those of skill in the art. For example, a composition,such as the expression construct or vector of the present invention, canstimulate upregulation of co-stimulatory molecules on antigen-presentingcells, induce nuclear translocation of NF-κB in antigen-presentingcells, activate toll-like receptors in antigen-presenting cells, orother activities involving cytokines or chemokines.

An amount of a composition that activates antigen-presenting cells which“enhances” an immune response refers to an amount in which an immuneresponse is observed that is greater or intensified or deviated in anyway with the addition of the composition when compared to the sameimmune response measured without the addition of the composition. Forexample, the lytic activity of cytotoxic T cells can be measured, e.g.,using a 51Cr release assay, with and without the composition. The amountof the substance at which the CTL lytic activity is enhanced as comparedto the CTL lytic activity without the composition is said to be anamount sufficient to enhance the immune response of the animal to theantigen. In a preferred embodiment, the immune response in enhanced by afactor of at least about 2, more preferably by a factor of about 3 ormore. The amount of cytokines secreted may also be altered.

The enhanced immune response may be an active or a passive immuneresponse. Alternatively, the response may be part of an adaptiveimmunotherapy approach in which antigen-presenting cells are obtainedwith from a subject (e.g., a patient), then transduced with acomposition comprising the expression vector or construct of the presentinvention. The antigen-presenting cells may be obtained from the bloodof the subject or bone marrow of the subject. In certain preferredembodiments, the antigen-presenting cells are isolated from the bonemarrow. In a preferred embodiment, the antigen-presenting cells areadministered to the same or different animal (e.g., same or differentdonors). In a preferred embodiment, the subject (e.g., a patient) has oris suspected of having a cancer, such as for example, prostate cancer,or has or is suspected of having an infectious disease. In otherembodiments the method of enhancing the immune response is practiced inconjunction with a known cancer therapy or any known therapy to treatthe infectious disease.

The expression construct, expression vector and/or transducedantigen-presenting cells can enhance or contribute to the effectivenessof a vaccine by, for example, enhancing the immunogenicity of weakerantigens such as highly purified or recombinant antigens, reducing theamount of antigen required for an immune response, reducing thefrequency of immunization required to provide protective immunity,improve the efficacy of vaccines in subjects with reduced or weakenedimmune responses, such as newborns, the aged, and immunocompromisedindividuals, and enhance the immunity at a target tissue, such asmucosal immunity, or promote cell-mediated or humoral immunity byeliciting a particular cytokine profile.

Yet further, an immunocompromised individual or subject is a subjectthat has a reduced or weakened immune response. Such individuals mayalso include a subject that has undergone chemotherapy or any othertherapy resulting in a weakened immune system, a transplant recipient, asubject currently taking immunosuppressants, an aging individual, or anyindividual that has a reduced and/or impaired CD4 T helper cells. It iscontemplated that the present invention can be utilize to enhance theamount and/or activity of CD4 T helper cells in an immunocompromisedsubject.

In specific embodiments, prior to administering the transducedantigen-presenting cell, the cells are challenged with antigens (alsoreferred herein as “target antigens”). After challenge, the transduced,loaded antigen-presenting cells are administered to the subjectparenterally, intradermally, intranodally, or intralymphatically.Additional parenteral routes include, but are not limited tosubcutaneous, intramuscular, intraperitoneal, intravenous,intraarterial, intramyocardial, transendocardial, transepicardial,intrathecal, and infusion techniques.

The target antigen, as used herein, is an antigen or immunologicalepitope on the antigen, which is crucial in immune recognition andultimate elimination or control of the disease-causing agent or diseasestate in a mammal. The immune recognition may be cellular and/orhumoral. In the case of intracellular pathogens and cancer, immunerecognition is preferably a T lymphocyte response.

The target antigen may be derived or isolated from a pathogenicmicroorganism such as viruses including HIV, (Korber et al, 1977)influenza, Herpes simplex, human papilloma virus (U.S. Pat. No.5,719,054), Hepatitis B (U.S. Pat. No. 5,780,036), Hepatitis C (U.S.Pat. No. 5,709,995), EBV, Cytomegalovirus (CMV) and the like. Targetantigen may be derived or isolated from pathogenic bacteria such as fromChlamydia (U.S. Pat. No. 5,869,608), Mycobacteria, Legionella,Meningiococcus, Group A Streptococcus, Salmonella, Listeria, Hemophilusinfluenzae (U.S. Pat. No. 5,955,596) and the like.

Target antigen may be derived or isolated from pathogenic yeastincluding Aspergillus, invasive Candida (U.S. Pat. No. 5,645,992),Nocardia, Histoplasmosis, Cryptosporidia and the like.

Target antigen may be derived or isolated from a pathogenic protozoanand pathogenic parasites including but not limited to Pneumocystiscarinii, Trypanosoma, Leishmania (U.S. Pat. No. 5,965,242), Plasmodium(U.S. Pat. No. 5,589,343) and Toxoplasma gondii.

Target antigen includes an antigen associated with a preneoplastic orhyperplastic state. Target antigen may also be associated with, orcausative of cancer. Such target antigen may be tumor specific antigen,tumor associated antigen (TAA) or tissue specific antigen, epitopethereof, and epitope agonist thereof. Such target antigens include butare not limited to carcinoembryonic antigen (CEA) and epitopes thereofsuch as CAP-1, CAP-1-6D (46) and the like (GenBank Accession No.M29540), MART-1 (Kawakami et al, 1994), MAGE-1 (U.S. Pat. No.5,750,395), MAGE-3, GAGE (U.S. Pat. No. 5,648,226), GP-100 (Kawakami etal., 1992), MUC-1, MUC-2, point mutated ras oncogene, normal and pointmutated p53 oncogenes (Hollstein et al., 1994), PSMA (Israeli et al.,1993), tyrosinase (Kwon et al. 1987) TRP-1 (gp75) (Cohen et al., 1990;U.S. Pat. No. 5,840,839), NY-ESO-1 (Chen et al., PNAS 1997), TRP-2(Jackson et al., 1992), TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2,(U.S. Pat. No. 5,550,214), BRC-I, BRC-II, bcr-abl, pax3-fkhr, ews-fli-1,modifications of TAAs and tissue specific antigen, splice variants ofTAAs, epitope agonists, and the like. Other TAAs may be identified,isolated and cloned by methods known in the art such as those disclosedin U.S. Pat. No. 4,514,506. Target antigen may also include one or moregrowth factors and splice variants of each.

An antigen may be expressed more frequently in cancer cells than innon-cancer cells. The antigen may result from contacting the modifieddendritic cell with prostate specific membrane antigen (PSMA) orfragment thereof. In certain embodiments, the modified dendritic cell iscontacted with a PSMA fragment having the amino acid sequence of SEQ IDNO: 4 (e.g., encoded by the nucleotide sequence of SEQ ID NO: 3).

For organisms that contain a DNA genome, a gene encoding a targetantigen or immunological epitope thereof of interest is isolated fromthe genomic DNA. For organisms with RNA genomes, the desired gene may beisolated from cDNA copies of the genome. If restriction maps of thegenome are available, the DNA fragment that contains the gene ofinterest is cleaved by restriction endonuclease digestion by methodsroutine in the art. In instances where the desired gene has beenpreviously cloned, the genes may be readily obtained from the availableclones. Alternatively, if the DNA sequence of the gene is known, thegene can be synthesized by any of the conventional techniques forsynthesis of deoxyribonucleic acids.

Genes encoding an antigen of interest can be amplified by cloning thegene into a bacterial host. For this purpose, various prokaryoticcloning vectors can be used. Examples are plasmids pBR322, pUC andpEMBL.

The genes encoding at least one target antigen or immunological epitopethereof can be prepared for insertion into the plasmid vectors designedfor recombination with a virus by standard techniques. In general, thecloned genes can be excised from the prokaryotic cloning vector byrestriction enzyme digestion. In most cases, the excised fragment willcontain the entire coding region of the gene. The DNA fragment carryingthe cloned gene can be modified as needed, for example, to make the endsof the fragment compatible with the insertion sites of the DNA vectorsused for recombination with a virus, then purified prior to insertioninto the vectors at restriction endonuclease cleavage sites (cloningsites).

Antigen loading of dendritic cells with antigens may be achieved byincubating dendritic cells or progenitor cells with the polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide. Antigens from cells or MHC molecules may be obtained byacid-elution or other methods known in the art (see Zitvogel et al.,1996).

One skilled in the art is fully aware that activation of theco-stimulatory molecule of the present invention relies uponoligomerization of ligand-binding domains, for example CID, to induceits activity. In specific embodiments, the ligand is a non-protein. Morespecifically, the ligand is a dimeric FK506 or dimeric FK506 analogs,which result in enhancement or positive regulation of the immuneresponse. The use of monomeric FK506 or monomeric FK506 analogs resultsin inhibition or reduction in the immune response negatively.

T-lymphocytes are activated by contact with the antigen-presenting cellthat comprises the expression vector of the present invention and hasbeen challenged, transfected, pulsed, or electrofused with an antigen.

Electrofusing in the present invention is a method of generating hybridcells. There are several advantages in producing cell hybrids byelectrofusion. For example, fusion parameters can be easily andaccurately electronically controlled to conditions depending on thecells to be fused. Further, electrofusion of cells has shown to theability to increase fusion efficiency over that of fusion by chemicalmeans or via biological fusogens. Electrofusion is performed by applyingelectric pulses to cells in suspension. By exposing cells to analternating electric field, cells are brought close to each other informing pearl chains in a process termed dielectrophoresis alignment.Subsequent higher voltage pulses cause cells to come into closercontact, reversible electropores are formed in reversibly permeabilizingand mechanically breaking down cell membranes, resulting in fusion.

T cells express a unique antigen binding receptor on their membrane(T-cell receptor), which can only recognize antigen in association withmajor histocompatibility complex (MHC) molecules on the surface of othercells. There are several populations of T cells, such as T helper cellsand T cytotoxic cells. T helper cells and T cytotoxic cells areprimarily distinguished by their display of the membrane boundglycoproteins CD4 and CD8, respectively. T helper cells secret variouslymphokines, that are crucial for the activation of B cells, T cytotoxiccells, macrophages and other cells of the immune system. In contrast, anaïve CD8 T cell that recognizes an antigen-MHC complex proliferates anddifferentiates into an effector cell called a cytotoxic CD8 T lymphocyte(CTL). CTLs eliminate cells of the body displaying antigen, such asvirus-infected cells and tumor cells, by producing substances thatresult in cell lysis.

CTL activity can be assessed by methods described herein or as would beknown to one of skill in the art. For example, CTLs may be assessed infreshly isolated peripheral blood mononuclear cells (PBMC), in aphytohaemaglutinin-stimulated IL-2 expanded cell line established fromPBMC (Bernard et al., 1998) or by T cells isolated from a previouslyimmunized subject and restimulated for 6 days with DC infected with anadenovirus vector containing antigen using standard 4 h 51Cr releasemicrotoxicity assays. One type of assay uses cloned T-cells. ClonedT-cells have been tested for their ability to mediate both perform andFas ligand-dependent killing in redirected cytotoxicity assays (Simpsonet al., 1998). The cloned cytotoxic T lymphocytes displayed both Fas-and perforin-dependent killing. Recently, an in vitro dehydrogenaserelease assay has been developed that takes advantage of a newfluorescent amplification system (Page et al., 1998). This approach issensitive, rapid, and reproducible and may be used advantageously formixed lymphocyte reaction (MLR). It may easily be further automated forlarge-scale cytotoxicity testing using cell membrane integrity, and isthus considered in the present invention. In another fluorometric assaydeveloped for detecting cell-mediated cytotoxicity, the fluorophore usedis the non-toxic molecule AlamarBlue (Nociari et al., 1998). TheAlamarBlue is fluorescently quenched (i.e., low quantum yield) untilmitochondrial reduction occurs, which then results in a dramaticincrease in the AlamarBlue fluorescence intensity (i.e., increase in thequantum yield). This assay is reported to be extremely sensitive,specific and requires a significantly lower number of effector cellsthan the standard 51 Cr release assay.

Other immune cells that are induced by the present invention includenatural killer cells (NK). NKs are lymphoid cells that lackantigen-specific receptors and are part of the innate immune system.Typically, infected cells are usually destroyed by T cells alerted byforeign particles bound the cell surface MHC. However, virus-infectedcells signal infection by expressing viral proteins that are recognizedby antibodies. These cells can be killed by NKs. In tumor cells, if thetumor cells lose expression of MHC I molecules, then it may besusceptible to NKs.

In further embodiments, the transduced antigen-presenting cell istransfected with tumor cell mRNA. The transduced transfectedantigen-presenting cell is administered to an animal to effect cytotoxicT lymphocytes and natural killer cell anti-tumor antigen immune responseand regulated using dimeric FK506 and dimeric FK506 analogs. The tumorcell mRNA is mRNA from a prostate tumor cell.

Yet further, the transduced antigen-presenting cell is pulsed with tumorcell lysates. The pulsed transduced antigen-presenting cells areadministered to an animal to effect cytotoxic T lymphocytes and naturalkiller cell anti-tumor antigen immune response and regulated usingdimeric FK506 and dimeric FK506 analogs. The tumor cell lysates is aprostate tumor cell lysate.

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference: Gossen and Bujard, Proc.Natl. Acad. Sci. USA, 89:5547-5551, 1992; Gossen et al., Science,268:1766-1769, 1995; Pelletier and Sonenberg, Nature, 334:320-325, 1988;Macejak and Sarnow, Nature, 353:90-94, 1991; Kageyama et al., (1987) J.Biol. Chem., 262,2345-2351; Arcone, et al., (1988) Nucl. Acids Res.,16(8), 3195-3207; Oliviero et al., (1987) EMBO J., 6, 1905-1912; Poliand Cortese, (1989) Proc. Nat'l Acad. Sci. USA, 86,8202-8206; Wilson etal., (1990) Mol. Cell. Biol., 6181-6191; Prowse and Baumann, (1988) MolCell Biol, 8,42-51; Ron, et al., (1991) Mol. Cell. Biol., 2887-2895;Potter et al., (1984) Proc. Nat'l Acad. Sci. USA, 81,7161-7165;Tur-Kaspa et al., (1986) Mol. Cell Biol., 6,716-718; Graham and van derEb, (1973) Virology, 52,456-467; Fechheimer et al., (1987) Proc. Nat'lAcad. Sci. 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VI. Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions-expression constructs, expressionvectors, fused proteins, transduced cells, activated DCs, transduced andloaded DCs—in a form appropriate for the intended application.Generally, this will entail preparing compositions that are essentiallyfree of pyrogens, as well as other impurities that could be harmful tohumans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. A pharmaceuticallyacceptable carrier includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well know in the art. Exceptinsofar as any conventional media or agent is incompatible with thevectors or cells of the present invention, its use in therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

For oral administration, the compositions of the present invention maybe incorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientalso may be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media, which can be employed, will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and general safety and purity standards as required by FDAOffice of Biologics standards.

VII. Methods for Treating a Disease

The present invention also encompasses methods of treatment orprevention of a disease caused by pathogenic microorganisms and/or ahyperproliferative disease.

Diseases may be treated or prevented by use of the present inventioninclude diseases caused by viruses, bacteria, yeast, parasites,protozoa, cancer cells and the like. The pharmaceutical composition ofthe present invention (transduced DCs, expression vector, expressionconstruct, etc.) of the present invention may be used as a generalizedimmune enhancer (DC activating composition or system) and as such hasutility in treating diseases. Exemplary disease that can be treatedand/or prevented utilizing the pharmaceutical composition of the presentinvention include, but are not limited to infections of viral etiologysuch as HIV, influenza, Herpes, viral hepatitis, Epstein Bar, polio,viral encephalitis, measles, chicken pox, Papilloma virus etc.; orinfections of bacterial etiology such as pneumonia, tuberculosis,syphilis, etc.; or infections of parasitic etiology such as malaria,trypanosomiasis, leishmaniasis, trichomoniasis, amoebiasis, etc.

Preneoplastic or hyperplastic states which may be treated or preventedusing the pharmaceutical composition of the present invention(transduced DCs, expression vector, expression construct, etc.) of thepresent invention include but are not limited to preneoplastic orhyperplastic states such as colon polyps, Crohn's disease, ulcerativecolitis, breast lesions and the like.

Cancers which may be treated using the pharmaceutical composition of thepresent invention of the present invention include, but are not limitedto primary or metastatic melanoma, adenocarcinoma, squamous cellcarcinoma, adenosquamous cell carcinoma, thymoma, lymphoma, sarcoma,lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma,leukemias, uterine cancer, breast cancer, prostate cancer, ovariancancer, pancreatic cancer, colon cancer, multiple myeloma,neuroblastoma, NPC, bladder cancer, cervical cancer and the like.

Other hyperproliferative diseases that may be treated using DCactivation system of the present invention include, but are not limitedto rheumatoid arthritis, inflammatory bowel disease, osteoarthritis,leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascularocclusion, restenosis, atherosclerosis, pre-neoplastic lesions (such asadenomatous hyperplasia and prostatic intraepithelial neoplasia),carcinoma in situ, oral hairy leukoplakia, or psoriasis.

In the method of treatment, the administration of the pharmaceuticalcomposition (expression construct, expression vector, fused protein,transduced cells, activated DCs, transduced and loaded DCs) of theinvention may be for either “prophylactic” or “therapeutic” purpose.When provided prophylactically, the pharmaceutical composition of thepresent invention is provided in advance of any symptom. Theprophylactic administration of pharmaceutical composition serves toprevent or ameliorate any subsequent infection or disease. When providedtherapeutically, the pharmaceutical composition is provided at or afterthe onset of a symptom of infection or disease. Thus the presentinvention may be provided either prior to the anticipated exposure to adisease-causing agent or disease state or after the initiation of theinfection or disease.

The term “unit dose” as it pertains to the inoculum refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of pharmaceutical compositioncalculated to produce the desired immunogenic effect in association withthe required diluent. The specifications for the novel unit dose of aninoculum of this invention are dictated by and are dependent upon theunique characteristics of the pharmaceutical composition and theparticular immunologic effect to be achieved.

An effective amount of the pharmaceutical composition would be theamount that achieves this selected result of enhancing the immuneresponse, and such an amount could be determined as a matter of routineby a person skilled in the art. For example, an effective amount of fortreating an immune system deficiency could be that amount necessary tocause activation of the immune system, resulting in the development ofan antigen specific immune response upon exposure to antigen. The termis also synonymous with “sufficient amount.”

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One of ordinary skillin the art can empirically determine the effective amount of aparticular composition of the present invention without necessitatingundue experimentation.

A. Genetic Based Therapies

Specifically, the present inventors intend to provide, to a cell, anexpression construct capable of providing a co-stimulatory polypeptide,such as CD40 to the cell, such as an antigen-presenting cell andactivating CD40. The lengthy discussion of expression vectors and thegenetic elements employed therein is incorporated into this section byreference. Particularly preferred expression vectors are viral vectorssuch as adenovirus, adeno-associated virus, herpes virus, vaccinia virusand retrovirus. Also preferred is lysosomal-encapsulated expressionvector.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10e4, 1×10e5, 1×10e6, 1×10e7,1×10e8, 1×10e9, 1×10e10, 1×10e 11 or 1×10e12 infectious particles to thepatient. Similar figures may be extrapolated for liposomal or othernon-viral formulations by comparing relative uptake efficiencies.Formulation as a pharmaceutically acceptable composition is discussedbelow.

B. Cell Based Therapy

Another therapy that is contemplated is the administration of transducedantigen-presenting cells. The antigen-presenting cells may be transducedin vitro. Formulation as a pharmaceutically acceptable composition isdiscussed above.

In cell based therapies, the transduced antigen-presenting cells may betransfected with target antigen nucleic acids, such as mRNA or DNA orproteins; pulsed with cell lysates, proteins or nucleic acids; orelectrofused with cells. The cells, proteins, cell lysates, or nucleicacid may derive from cells, such as tumor cells or other pathogenicmicroorganism, for example, viruses, bacteria, protozoa, etc.

C. Combination Therapies

In order to increase the effectiveness of the expression vector of thepresent invention, it may be desirable to combine these compositions andmethods of the invention with an agent effective in the treatment of thedisease.

In certain embodiments, anti-cancer agents may be used in combinationwith the present invention. An “anti-cancer” agent is capable ofnegatively affecting cancer in a subject, for example, by killing one ormore cancer cells, inducing apoptosis in one or more cancer cells,reducing the growth rate of one or more cancer cells, reducing theincidence or number of metastases, reducing a tumor's size, inhibiting atumor's growth, reducing the blood supply to a tumor or one or morecancer cells, promoting an immune response against one or more cancercells or a tumor, preventing or inhibiting the progression of a cancer,or increasing the lifespan of a subject with a cancer. Anti-canceragents include, for example, chemotherapy agents (chemotherapy),radiotherapy agents (radiotherapy), a surgical procedure (surgery),immune therapy agents (immunotherapy), genetic therapy agents (genetherapy), hormonal therapy, other biological agents (biotherapy) and/oralternative therapies.

In further embodiments antibiotics can be used in combination with thepharmaceutical composition of the present invention to treat and/orprevent an infectious disease. Such antibiotics include, but are notlimited to, amikacin, aminoglycosides (e.g., gentamycin), amoxicillin,amphotericin B, ampicillin, antimonials, atovaquone sodiumstibogluconate, azithromycin, capreomycin, cefotaxime, cefoxitin,ceftriaxone, chloramphenicol, clarithromycin, clindamycin, clofazimine,cycloserine, dapsone, doxycycline, ethambutol, ethionamide, fluconazole,fluoroquinolones, isoniazid, itraconazole, kanamycin, ketoconazole,minocycline, ofloxacin), para-aminosalicylic acid, pentamidine,polymixin definsins, prothionamide, pyrazinamide, pyrimethaminesulfadiazine, quinolones (e.g., ciprofloxacin), rifabutin, rifampin,sparfloxacin, streptomycin, sulfonamides, tetracyclines, thiacetazone,trimethaprim-sulfamethoxazole, viomycin or combinations thereof.

More generally, such an agent would be provided in a combined amountwith the expression vector effective to kill or inhibit proliferation ofa cancer cell and/or microorganism. This process may involve contactingthe cell(s) with an agent(s) and the pharmaceutical composition of thepresent invention at the same time or within a period of time whereinseparate administration of the pharmaceutical composition of the presentinvention and an agent to a cell, tissue or organism produces a desiredtherapeutic benefit. This may be achieved by contacting the cell, tissueor organism with a single composition or pharmacological formulationthat includes both the pharmaceutical composition of the presentinvention and one or more agents, or by contacting the cell with two ormore distinct compositions or formulations, wherein one compositionincludes the pharmaceutical composition of the present invention and theother includes one or more agents.

The terms “contacted” and “exposed,” when applied to a cell, tissue ororganism, are used herein to describe the process by which thepharmaceutical composition and/or another agent, such as for example achemotherapeutic or radiotherapeutic agent, are delivered to a targetcell, tissue or organism or are placed in direct juxtaposition with thetarget cell, tissue or organism. To achieve cell killing or stasis, thepharmaceutical composition and/or additional agent(s) are delivered toone or more cells in a combined amount effective to kill the cell(s) orprevent them from dividing

The administration of the pharmaceutical composition may precede, beco-current with and/or follow the other agent(s) by intervals rangingfrom minutes to weeks. In embodiments where the pharmaceuticalcomposition of the present invention, and other agent(s) are appliedseparately to a cell, tissue or organism, one would generally ensurethat a significant period of time did not expire between the times ofeach delivery, such that the pharmaceutical composition of the presentinvention and agent(s) would still be able to exert an advantageouslycombined effect on the cell, tissue or organism. For example, in suchinstances, it is contemplated that one may contact the cell, tissue ororganism with two, three, four or more modalities substantiallysimultaneously (i.e., within less than about a minute) as thepharmaceutical composition of the present invention. In other aspects,one or more agents may be administered within of from substantiallysimultaneously, about 1 minute, to about 24 hours to about 7 days toabout 1 to about 8 weeks or more, and any range derivable therein, priorto and/or after administering the expression vector. Yet further,various combination regimens of the pharmaceutical composition of thepresent invention and one or more agents may be employed.

EXAMPLES

The following examples are provided to illustrate aspects of theinvention and are not limiting.

Example 1: Materials and Methods

Described hereafter are materials and methods utilized in studiesdescribed in subsequent Examples.

Tumor Cell Lines and Peptides

NA-6-Mel, T2, SK-Mel-37 and LNCaP cell lines were purchased from ATCC(Manassas, VA). HLA-A2-restricted peptides MAGE-3 p271-279 (FLWGPRALV)(SEQ ID NO: 8), influenza matrix (IM) p58-66 (GILGFVFTL) (SEQ ID NO: 9),and HIV-1 gag p77-85 (SLYNTVATL) (SEQ ID NO: 10) were used to analyzeCD8+ T cell responses. In T helper cell polarization experiments,tetanus toxoid peptide TTp30 FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 5) wasused. All peptides were synthesized by Genemed Synthesis Inc (SanFrancisco, CA), with an HPLC-determined purity of >95%.

Recombinant Adenovirus Encoding Human Inducible CD40

The human CD40 cytoplasmic domain was Pfu I polymerase (Stratagene, LaJolla, California) amplified from human monocyte-derived DC cDNA usingan Xho I-flanked 5′ primer (5hCD40X),5′-atatactcgagaaaaaggtggccaagaagccaacc-3′ (SEQ ID NO: 11), and a SalI-flanked 3′ primer (3hCD40S),5′-atatagtcgactcactgtctctcctgcactgagatg-3′ (SEQ ID NO: 12). The PCRfragment was subcloned into Sal I-digested pSH1/M-FvFvls-E15 andsequenced to create pSH1/M-FvFvls-CD40-E. Inducible CD40 wassubsequently subcloned into a non-replicating E1, E3-deletedAd5/f35-based vector expressing the transgene under a cytomegalovirusearly/immediate promoter. The iCD40-encoding sequence was confirmed byrestriction digest and sequencing. Amplification, purification, andtitration of all adenoviruses were carried out in the Viral Vector CoreFacility of Baylor College of Medicine.

Western Blot

Total cellular extracts were prepared with RIPA buffer containing aprotease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) andquantitated using a detergent-compatible protein concentration assay(Bio-Rad, Hercules, CA). 10-15 micrograms of total protein wereroutinely separated on 12% SDS-PAGE gels, and proteins were transferredto nitrocellulose membranes (Bio-Rad). Blots were hybridized with goatanti-CD40 (T-20, Santa Cruz Biotechnology, Santa Cruz, CA) and mouseanti-alpha-tubulin (Santa Cruz Biotechnology) Abs followed by donkeyanti-goat and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology),respectively. Blots were developed using the SuperSignal West DuraStable substrate system (Pierce, Rockford, Illinois).

Generation and Stimulation of Human DCs

Peripheral blood mononuclear cells (PBMCs) from healthy donors wereisolated by density centrifugation of heparinized blood on Lymphoprep(Nycomed, Oslo, Norway). PBMCs were washed with PBS, resuspended inCellGenix DC medium (Freiburg, Germany) and allowed to adhere in cultureplates for 2 h at 37° C. and 5% CO₂. Nonadherent cells were removed byextensive washings, and adherent monocytes were cultured for 5 days inthe presence of 500 U/ml hIL-4 and 800 U/ml hGM-CSF (R&D Systems,Minneapolis, MN). As assessed by morphology and FACS analysis, theresulting immature DCs (imDCs) were MHC-class I, IIhi, and expressedCD40lo, CD80lo, CD83lo, CD86lo. The imDCs were CD14neg and contained <3%of contaminating CD3+ T, CD19+B, and CD16+ NK cells.

Approximately 2×10⁶ cells/ml were cultured in a 24-well dish andtransduced with adenoviruses at 10,000 viral particle (vp)/cell (˜160MOI) for 90 min at 37° C. and 5% CO₂. Immediately after transduction DCswere stimulated with MPL, FSL-1, Pam3CSK4 (InvivoGen, San Diego, CA),LPS (Sigma-Aldrich, St. Louis, MO), AP20187 (kind gift from ARIADPharmaceuticals, Cambridge, MA) or maturation cocktail (MC), containing10 ng/ml TNF-alpha, 10 ng/ml IL-1beta, 150 ng/ml IL-6 (R&D Systems,Minneapolis, MN) and 1 micrograms/ml of PGE₂ (Cayman Chemicals, AnnArbor, MI). In T cell assays DCs were pulsed with 50 micrograms/ml ofPSMA or MAGE 3 peptide 24 hours before and after adenoviraltransduction.

Surface Markers and Cytokine Production

Cell surface staining was done with fluorochrome-conjugated monoclonalantibodies (BD Biosciences, San Diego, CA). Cells were analyzed on aFACSCalibur cytometer (BD Biosciences, San Jose, CA). Cytokines weremeasured in culture supernatants using enzyme-linked immunosorbent assaykits for human IL-6 and IL-12p70 (BD Biosciences).

Real Time Q-PCR Assay for Human SOCS1

Total RNA was purified and reverse transcribed with random hexamersusing SuperScript II RTase (Invitrogen, Carlsbad, CA). mRNA levels werequantified in DCs by subjecting cDNA to TaqMan PCR analysis using theGeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City,CA). Pre-developed sequence detection reagents (Applied Biosystems)specific for human SOCS1 and 18S rRNA, including forward and reverseprimers as well as a fluorogenic TaqMan FAM-labeled hybridization probe,were supplied as mixtures and were used at 1 microliter/20 microliterPCR. Samples were run in duplicates. The level of SOCS1 expression ineach sample was normalized to the level of 18S rRNA from the same sampleusing the comparative 2-ΔΔCT method²¹.

DC Migration Assay

Chemotaxis of DCs was measured by migration through a polycarbonatefilter with 8 micrometer pore size in 96-Multiwell HTS Fluoroblok plates(BD Biosciences). Assay medium (250 μL) containing 100 ng/ml CCL19 (R&DSystems) or assay medium alone (as a control for spontaneous migration)were loaded into the lower chamber. DCs (50,000) were labeled withGreen-CMFDA cell tracker (Invitrogen), unstimulated or stimulated for 48h with the indicated reagents, and were added to the upper chamber in atotal volume of 50 μL for 1 hour at 37° C. and 5% CO₂. Fluorescence ofcells, which had migrated through the microporous membrane, was measuredusing the FLUOstar OPTIMA reader (BMG Labtech Inc., Durham, NC). Themean fluorescence of spontaneously migrated cells was subtracted fromthe total number of migrated cells for each condition.

IFN-Gamma ELISPOT Assay

DCs from HLA-A2-positive healthy volunteers were pulsed with MAGE-3 A2.1peptide (residues 271-279; FLWGPRALV) (SEQ ID NO: 8) on day 4 ofculture, followed by transduction with Ad-iCD40 and stimulation withvarious stimuli on day 5. Autologous T cells were purified from PBMCs bynegative selection (Miltenyi Biotec, Auburn, CA) and mixed with DCs atDC:T cell ratio 1:3. Cells were incubated in complete RPMI with 20 U/mlhIL-2 (R&D Systems) and 25 micrograms/ml of MAGE 3 A2.1 peptide. T cellswere restimulated at day 7 and assayed at day 14 of culture.

ELISPOT Quantitation

Flat-bottom, 96-well nitrocellulose plates (MultiScreen-HA; Millipore,Bedford, MA) were coated with IFN-gamma mAb (2 μg/ml, 1-D1K; Mabtech,Stockholm, Sweden) and incubated overnight at 4° C. After washings withPBS containing 0.05% TWEEN 20, plates were blocked with complete RPMIfor 2 h at 37° C. A total of 1×10 5 presensitized CD8+ T effector cellswere added to each well and incubated for 20 h with 25 micrograms/mlpeptides. Plates were then washed thoroughly with PBS containing 0.05%Tween 20, and anti-IFN-mAb (0.2 μg/ml, 7-B6-1-biotin; Mabtech) was addedto each well. After incubation for 2 h at 37° C., plates were washed anddeveloped with streptavidin-alkaline phosphatase (1 μg/ml; Mabtech) for1 h at room temperature. After washing, substrate(3-amino-9-ethyl-carbazole; Sigma-Aldrich) was added and incubated for 5min. Plate membranes displayed dark-pink spots that were scanned andanalyzed by ZellNet Consulting Inc. (Fort Lee, NJ).

Chromium Release Assay

Antigen recognition was assessed using target cells labeled with 51Chromium (Amersham) for 1 h at 37° C. and washed three times. Labeledtarget cells (5000 cells in 50 μl) were then added to effector cells(100 μl) at the indicated effector:target cell ratios in V-bottommicrowell plates at the indicated concentrations. Supernatants wereharvested after 6-h incubation at 37° C., and chromium release wasmeasured using MicroBeta Trilux counter (Perkin-Elmer Inc, Torrance CA).Assays involving LNCaP cells were run for 18 hours. The percentage ofspecific lysis was calculated as: 100*[(experimental−spontaneousrelease)/(maximum−spontaneous release)].

Tetramer Staining

HLA-A2 tetramers assembled with MAGE-3.A2 peptide (FLWGPRALV) (SEQ IDNO: 8) were obtained from Baylor College of Medicine Tetramer CoreFacility (Houston, TX). Presensitized CD8+ T cells in 50 μl of PBScontaining 0.5% FCS were stained with PE-labeled tetramer for 15 min onice before addition of FITC-CD8 mAb (BD Biosciences). After washing,results were analyzed by flow cytometry.

Polarization of Naïve T Helper Cells

Naïve CD4+CD45RA+ T-cells from HLA-DR11.5-positive donors (genotypedusing FASTYPE HLA-DNA SSP typing kit; BioSynthesis, Lewisville, TX) wereisolated by negative selection using naïve CD4+ T cell isolation kit(Miltenyi Biotec, Auburn, CA). T cells were stimulated with autologousDCs pulsed with tetanus toxoid (5 FU/ml) and stimulated with variousstimuli at a stimulator to responder ratio of 1:10. After 7 days, Tcells were restimulated with autologous DCs pulsed with theHLA-DR11.5-restricted helper peptide TTp30 and transduced withadenovector Ad-iCD40. Cells were stained with PE-anti-CD4 Ab (BDBiosciences), fixed and permeabilized using BD Cytofix/Cytoperm kit (BDBiosciences), then stained with hIFN-gamma mAb (eBioscience, San Diego,CA) and analyzed by flow cytometry. Supernatants were analyzed usinghuman TH1/TH2 BD Cytometric Bead Array Flex Set on BD FACSArrayBioanalyzer (BD Biosciences).

PSMA Protein Purification

The baculovirus transfer vector, pAcGP67A (BD Biosciences) containingthe cDNA of extracellular portion of PSMA (residues 44-750) was kindlyprovided by Dr Pamela J. Bjorkman (Howard Hughes Medical Institute,California Institute of Technology, Pasadena, CA). PSMA was fused with ahydrophobic secretion signal, Factor Xa cleavage site, and N-terminal6x-His (SEQ ID NO: 37) affinity tag. High titer baculovirus was producedby the Baculovirus/Monoclonal antibody core facility of Baylor Collegeof Medicine. PSMA protein was produced in High 5 cells infected withrecombinant virus, and protein was purified from cell supernatants usingNi-NTA affinity columns (Qiagen, Chatsworth, CA) as previously described24. After purification the ˜100 kDa solitary band of PSMA protein wasdetected by silver staining of acrylamide gels.

Migration of Human DCs in Mouse Host

In order to assess the migration of human DCs in vivo, adenovector,Ad5-CBR, which expresses red-shifted (emission peak=613 nM) luciferasefrom Pyrophorus plagiophalamus click beetles (Promega, Madison, WI) wasdeveloped. Human DCs were transduced with ˜50 MOI of Ad5-CBR, and 160MOI of Ad5f35-iCD40. DCs were then matured with MC or 1 micrograms/mlLPS (Sigma-Aldrich, St. Louis, MO). Mouse bone-marrow derived DCs wereobtained as described before¹³ and were matured with 1 micrograms/mlLPS. Approximately 2×10⁶ DCs were injected into the left and right hindfootpads of irradiated (250 rads) Balb/c mice (both hind legs of threemice per group, n=6). Mice were i.p. injected with D-Luciferin (˜1 mg/25g animal) and imaged over several days using an IVIS™ 100 imaging system(Xenogen Corp., Alameda, CA). Luminescent signal was measured in 3 miceper group, and popliteal and inguinal lymph nodes (LN) were removed atday 2 post-DC inoculation. The LNs' signal was measured and thebackground was subtracted for each group (n=6).

Data Analysis

Results are expressed as the mean±standard error. Sample size wasdetermined with a power of 0.8, with a one-sided alpha-level of 0.05.Differences between experimental groups were determined by the Student ttest.

Example 2: Expression of iCD40 and Induction of DC Maturation

To investigate whether iCD40 signaling can enhance the immunogenicfunctions of human DCs, adenovirus, Ad5/f35-ihCD40 (simplified toAd-iCD40) was generated, expressing inducible human CD40 receptor, basedon the previously described mouse iCD40 vectorl3 (FIG. 1A). The humanCD40 cytoplasmic signaling domain was cloned downstream of amyristoylation-targeting domain and two tandem domains (from humanFKBP12(V36), designated as “Fv”), which bind dimerizing drug AP2018722.As shown in FIG. 1B, immature DCs expressed endogenous CD40, which wasinduced by LPS and CD40L. Transduction of Ad-iCD40 led to expression ofthe distinctly sized iCD40, which did not interfere with endogenous CD40expression. Interestingly, the expression of iCD40 was alsosignificantly enhanced by LPS stimulation, likely due to inducibility ofubiquitous transcription factors binding the “constitutive” CMVpromoter.

One of the issues for the design of DC-based vaccines is to obtain fullymatured and activated DCs, as maturation status is linked to thetransition from a tolerogenic to an activating, immunogenicstate^(4,13,23.)It has been shown that expression of mouse variantAd-iCD40 can induce murine bone marrow-derived DC maturation¹³. Todetermine whether humanized iCD40 affects the expression of maturationmarkers in DCs, DCs were transduced with Ad-iCD40 and the expression ofmaturation markers CD40, CD80, CD83, and CD86 were evaluated. TLR-4signaling mediated by LPS or its derivative MPL is a potent inducer ofDC maturation^(18,24-26). It was also previously reported thatendogenous CD40 signaling specifically up-regulates CD83 expression inhuman DCs27. Consistent with previous reports²⁷, the expression levelsof CD83 were upregulated upon Ad-iCD40 transduction, and CD83 expressionwas further upregulated following LPS or MPL addition (FIG. 2 and datanot shown). As shown in FIG. 2 the expression of CD40, CD80, and CD86maturation markers were also highly induced by iCD40 with the additionof the dimerizer, AP20187, and further induced by the addition of LPS orMPL. In contrast, control adenovirus expressing renilla luciferase,Ad-Luc, only provided incidental activation. These results show that thecombination of inducible CD40 and TLR-4 ligands provides sufficientstimulation for full DC maturation. This up-regulation of CD40, CD80,CD83 and CD86 expression was similar to that achieved by standardmaturation cocktail (MC) and CD40L (data not shown).

Example 3: Inducible CD40 Signaling and TLR4 Ligation Synergize forIL-12p70 and L-6 Production in Human DCs

Interleukin-12 (IL-12) activates T and NK cell responses, and inducesIFN-gamma production. It also favors the differentiation of TH1 cellsand is a vital link between innate and adaptive immunity^(1,28).Therefore, induction of biologically active IL-12p70 heterodimer islikely critical for optimum DC-based vaccines. Nonetheless, current DCvaccination protocols that include PGE₂ produce only limited IL-1229.IL-12 is a heterodimeric cytokine consisting of p40 and p35 chains.Previously, it was reported that inducible CD40 signaling promotes theexpression of the p35 subunit of IL-12p70 in mouse bone marrow-derivedDCs¹³. It was also reported that TLR-4 ligation can promote p40expression³⁰. Therefore, iCD40-DCs was cultured in the presence of LPSor MPL and assayed supernatants by ELISA for production of IL-12p70.

Predictably, similar to DCs treated with standard MC, iCD40-DCs did notproduce detectable IL-12p70 heterodimer (FIG. 3A). If PGE₂ was withheldfrom the MC, DCs produced detectable but low levels of IL-12p70 (FIG.3B), consistent with a potentially deleterious role for PGE₂.Furthermore, DCs cultured for 12 h in the presence of LPS or MPL alonealso failed to produce IL-12 (<30 pg/ml). However, whenAd-iCD40-transduced DCs were cultured in the presence of either MPL orLPS they produced very high levels of IL-12p70 (16.4±7.8 ng/ml for MPL).This level of IL-12 was about 25-fold higher than levels induced bystandard MC lacking PGE₂. Interestingly, this synergism of iCD40 andTLR4 was partially independent of AP20187 addition, implying that basaliCD40 signaling can also synergize with TLR4 ligation. IL-12p70production in iCD40-DCs was also dose-dependent as IL-12 levelscorrelated with viral particles dose (Supplementary FIG. 1 ).

To determine whether other TLRs could also synergize with iCD40, ligandsfor TLR 1,2,4, and 6 were tested for IL12 production. As shown in FIG.3B, FSL-1 (ligand for TLR2/TLR6) and Pam3 CSK4 (ligand for TLR1/TLR2)induced only low levels of IL12-p70 in iCD40-DCs. As before, TLR4ligation with MPL or LPS synergized with CD40 signaling.

Since CD40 signaling is normally tightly restricted to a relativelyshort time period^(31,32) potentially limiting adaptive immunity, it wasdetermined whether iCD40 could induce not only enhanced, but alsoprolonged, expression of IL-12p70 in TLR-4-stimulated DCs. To evaluatethe kinetics of IL-12 expression, LPS-treated iCD40-DCs with LPS andCD40L-stimulated DCs were compared. It was observed that iCD40-DCs wereable to produce IL-12p70 for over 72 hours post stimulation compared toCD40L or control vector-transduced DCs (FIG. 3C) in which IL-12p70expression ceased when LPS stimulation was removed. These resultsindicate that inducible CD40 signaling allows DCs to produce increasedlevels of IL-12p70 continuously in response to TLR-4 stimulation.

Finally, the induction of the suppressor of cytokine signaling (SOCS1)was evaluated. SOCS1 is negative feedback inhibitor of DC activation,that can attenuate³³ responsiveness to LPS and cytokine stimulation³⁴.FIG. 3D shows that LPS stimulation up-regulated SOCS1 expression in DCs,as previously reported³³. Strikingly, however, in the presence of LPS,iCD40-DCs expressed 3-fold lower levels of SOCS1 than CD40L-stimulatedDCs. Moreover, iCD40 did not induce SOCS1 by itself, unlike CD40L. Thesedata indicate that iCD40 can partially bypass SOCS1 induction in humanDCs and may partly explain the observed sustained elevation of IL-12levels and DC maturation markers.

In addition to IL-12, IL-6 plays an important role in cell survival andresistance to T regulatory cells^(19,35). It was observed that upontransfection with Ad-iCD40, IL-6 expression was significantly enhancedand further upregulated when iCD40-DCs were stimulated with dimerizerdrug and TLR-4 ligands (FIG. 3E). Thus, iCD40 signaling is sufficientfor production of some pro-inflammatory cytokines, but requiresadditional TLR signaling for production of the key TH1 cytokine, IL-12.

Example 4: iCD40 and TLR-4-Stimulated DCs Enhance Antigen-Specific TH1Polarization

To further investigate whether iCD40-DCs matured with TLR-4 ligands caneffectively prime CD4+ T helper (TH) cells, it was determined whetherthey can augment CD4+epitope-specific T-cell responses in vitro. NaïveCD4+CD45RA+ T cells were stimulated for 7 days in the presence ofautologous Ad-iCD40 DCs pulsed with the model antigen, tetanus toxoid.At day 7, T cells were stimulated with the MHC class II-restrictedtetanus toxoid epitope, TTp30. FIG. 4A shows that the production ofIFN-gamma was significantly increased in the CD4+ T cells co-culturedwith iCD40-DCs and iCD40-DCs stimulated with either MPL or MC. IFN-gammaproduction was iCD40-specific, as it was not induced by control virusAd-GFP-transduced DCs or by MPL or MC stimulation alone (FIG. 4A anddata not shown). In addition, T cell polarization was analyzed byassessing TH1/TH2 cytokine levels in the supernatants of T cells using acytometric bead array (FIG. 4B). The levels of IFN-gamma, TNF-alpha,IL-4, and IL-5 secreted cytokines were increased in helper T cellsstimulated by iCD40-DCs, indicating the expansion of both TH1 andTH2-polarized T cells. However, the levels of TH1 cytokines weresignificantly higher than TH2-associated cytokines, indicating apredominant expansion of TH1 cells. In contrast, induction ofTT-specific CD4+ T-helper cells from naïve CD4+CD45RA+ cells, usingMC-matured DCs, led to only a modest bias in TT epitope-specific TH1differentiation. These results suggest that iCD40 signaling in DCsenables them to effectively induce antigen-specific TH1 differentiation,possibly due to higher IL-12 production.

Example 5: MPL-Stimulated iCD40-DCs Induce Strong Tumor Antigen-SpecificCTL Responses

It was determined whether iCD40 and MPL could enhance cytotoxic Tlymphocyte (CTL) responses to poorly immunogenic melanoma self-antigenMAGE-3. iCD40-DCs from HLA-A2-positive donors were pulsed with class-IHLA-A2.1-restricted MAGE3-derived immunodominant peptide, FLWGPRALV (SEQID NO: 8), and co-cultured with autologous T cells. After a series ofstimulations, the frequency of antigen-specific T cells was assessed byIFN-gamma-specific ELISPOT assay (FIG. 5A). iCD40-DCs stimulated withMPL led to a 50% increase in MAGE-3-specific T cells relative toiCD40-DCs stimulated with MC and about a five-fold increase inantigen-specific T cells compared to control non-transduced (WT) DCs.

It also was determined whether iCD40-DCs were capable of enhancingCTL-mediated killing of tumor cells in an antigen-specific fashion.Immature DCs from HLA-A2-positive volunteers were transfected withAd-iCD40, pulsed with MAGE-3 protein, and used as stimulators togenerate CTLs in vitro. SK-MEL-37 cells (HLA-A2+, MAGE-3+) and T2 cellspulsed with MAGE-3 A2.1 peptide (HLA-A2+, MAGE-3+) were utilized astargets. NA-6-MEL cells (HLA A2−, MAGE-3+) and T2 cells (HLA-A2+) pulsedwith an irrelevant A2.1-restricted influenza matrix peptide served asnegative controls. As shown in FIG. 5B, the CTLs induced by iCD40-DCswere capable of efficiently recognizing and lysing their cognate targets(SK-MEL-37, left top panel), and also T2 cells pulsed with MAGE-3 A2.1peptide (lower left panel), indicating the presence of MAGE-3-specificCTLs. In contrast, control targets were lysed at significantly lowerlevels (right panels). Improved lytic activity was consistently observedwhen iCD40-DCs treated with MPL or MC were used as stimulators comparedwith non-transduced DCs treated with MPL or MC alone. In addition, asignificant expansion of MAGE-3/HLA-A2-specific tetramer positiveCD8+CTLs by iCD40-DCs that were treated with MPL (FIG. 5C) was observed.

Similarly, to test whether LPS and iCD40-stimulated DCs could enhanceCTL lytic activity, their ability to break tolerance toprostate-specific membrane antigen (PSMA) was examined. DCs generatedfrom healthy HLA-A2+ volunteers were pulsed with PSMA protein³⁶ orMAGE-3, transduced with AD-iCD40 or Ad-Luc, and were co-cultured withautologous T cells. After three rounds of stimulation, antigen-specificCTL activity was measured by chromium-release assay using LNCaP cells(HLA−A2+PSMA+) as targets and SK-Mel-37 (HLA−A2+PSMA−) as control cellsfor PSMA-pulsed DCs (FIG. 6A). SK-Mel-37 cells (MAGE-3+) were used astargets when DCs of the same donor were pulsed with MAGE-3, and LNCaPcells (MAGE-3−) were used here as negative controls (FIG. 6B).Collectively, these data indicate that iCD40-transduced DCs are capableof inducing significantly more potent antigen-specific CTL responses invitro than MC-treated DCs.

Example 6: Inducible CD40 Enhances CCR7 Expression and MigratoryAbilities of DCs without PGE₂

In addition to other maturation markers, CCR7 is up-regulated on DCsupon maturation and is responsible for directing their migration todraining lymph nodes³⁷. Recently, several reports have indicated that,apart from chemotaxis, CCR7 also affects DC “cytoarchitecture”, the rateof endocytosis, survival, migratory speed, and maturation³⁸. Along withcostimulatory molecules and TH1 cytokines, iCD40 specificallyup-regulates CCR7 expression in human DCs (FIG. 7A). Moreover, CCR7expression correlated with Ad-iCD40 viral dose-escalation (SupplementaryFIG. 2 ).

Because CCR7 expression levels correlate with enhanced migration towardsMIP-3 beta CCL19), it was determined whether human iCD40-DCs couldmigrate in vitro towards MIP-3 beta in transwell assays. FIG. 7B showsthat iCD40-DCs treated with AP20187 dimerizer has migration levelscomparable to those induced by MC. Moreover, iCD40-DC migration wasfurther increased by MPL or MC stimulation, even when PGE₂ was notpresent. These data were highly reproducible and indicate that iCD40 issufficient to induce CCR7 expression and DC migration in vitro incontrast to the widely held belief that PGE₂ is essential for lymph nodehoming of human DC.

Chemokines and chemokine receptors share a high degree of sequenceidentity within a species and between species³⁹. On the basis of thisknowledge, a novel xenograft model was developed for monitoring themigration of human DCs in vivo. Human DCs were transduced with iCD40 andmatured with LPS or MC, and mouse DCs were matured with LPS. Since DCswere co-transduced with Ad5-CBR, bioluminescence was immediately visible(FIG. 7C and not shown). As expected, immature DCs did not migrate tothe draining popliteal lymph nodes. However, iCD40-DCs matured with LPSor MC were detectable in the xenogeneic popliteal lymph nodes within 2days post-inoculation (FIG. 7C). The migration of iCD40-DCs stimulatedwith LPS was significantly (p=0.036) higher than non-stimulated DCs andwas comparable to mouse DC migration (FIG. 7D). Moreover, at day 2 theiCD40-DCs were detected in inguinal LNs while MC-stimulated DCs wereundetectable, suggesting higher migratory abilities of iCD40-DCs thanstimulated with MC. Collectively, these results indicate that iCD40signaling in DCs plays a critical role in controlling CCR7 expressionand is sufficient for DC migration to lymph nodes. The migration ofiCD40-DCs is further enhanced when the cells are stimulated with LPS,correlating with enhanced CCR7 expression.

Example 7: Analysis of Results Presented in Example 2 to Example 6 andDocuments Cited in Specification Through Example 7

Dendritic cell efficacy depends on many variables, especially maturationstatus and efficient migration to lymph nodes. Several clinical trialsin cancer patients showed the potency of DCs to induce adaptive immunityto tumor-specific antigens^(40,41,42). However, clinical responses weretransient, and warrant further improvement in DC vaccine design^(43,44).Limitation of current DC-based vaccines are the transient activationstate within lymphoid tissues, low induction of CD4+ T cell immunity,and impaired ability to migrate to the draining lymph nodes⁴⁵. Less than24 hours following exposure to LPS, DCs terminate synthesis of theTH1-polarizing cytokine, IL-12, and become refractory to furtherstimuli⁴⁶, limiting their ability to activate T helper cells and CTLs.Other studies indicate that less than 5% of intradermally administeredmature DCs reach the lymph nodes, showing inefficient homing³⁹. Thesefindings underscore the need for either prolonging the activation stateand migratory capacities of the DCs and/or temporally coordinating theDC activation “window” with engagement of cognate T cells within lymphnodes.

A method for promoting mouse DC function in vivo was developed bymanipulation of a chimeric inducible CD40 receptor¹³. It has beenobserved that the inducible CD40 approach is also effective in enhancingthe immunostimulatory function of human DCs. Consistent with previousreports of the synergistic activity of combining TLR and CD40 signalingfor IL-12p70 secretion, iCD40 plus TLR4 signaling induced high levelIL-12 secretion, DC maturation, T cell stimulatory functions, andextensive migratory capacities^(20,47).

It was also demonstrated that increased and prolonged secretion ofIL-12p70 in DCs could break self tolerance, which likely is attributablein part to over-riding the production of SOCS1, which inhibits IL-12signaling³⁴. It has been determined that although endogenous CD40signaling stimulated by soluble CD40L leads to SOCS1 upregulation, iCD40activates DCs without significant SOCS1 induction. Additionally, iCD40signaling unleashes high and prolonged expression of IL-12p70 in DCs,which exhibit enhanced potency in stimulating CD4+ T cells and CTLs.

IL-6 is implicated in the survival of many different cell types byactivation of anti-apoptotic pathways, such as p38 MAPK, ERK1, 2 47 andPI3-kinase⁴⁸. The induction of IL-6 expression by iCD40 and TLR-4signalings in DCs also was identified. This finding could partly explainthe prolonged survival of DCs described previously¹³. Furthermore, IL-6expression is critical in the ability of DCs to inhibit the generationof CD4+CD25+ T regulatory cells³⁵. In this context, an iCD40-DCs-basedvaccine could potentially suppress negative regulators in vivo,inhibiting peripheral tolerance to targeting antigens.

One major focus of cancer immunotherapy has been the design of vaccinesto promote strong tumor antigen-specific CTL responses in cancerpatients3. However, accumulating evidence suggests that CD4+ T cellsalso play a critical role in antitumor immunity, as they contribute tothe induction, persistence and expansion of CD8+ T cells⁴⁹. Our studyshowed that iCD40-DCs could effectively prime naïve T cells andeffectively expand antigen-specific cells representing both arms of theimmune response (i.e. MAGE-3 and PSMA specific CTLs and TT-specific CD4+T cells). It was demonstrated that TH1 (IFN-gamma and TNF-alpha)cytokines were produced predominantly in the milieu ofiCD40-DC-stimulated CD4+ T cells, indicating expansion of TH1 cells. Asexpected, these cytokines were not detected when T cells were stimulatedwith MC-treated DCs, because PGE₂ (a key MC component) is a powerfulsuppressor of TH1 responses^(50,51,52.)

Recent mouse studies have shown that DC migration directly correlateswith T cell proliferation⁵³. Therefore, the increase in migration shouldenhance efficacy of DC-based vaccines⁴⁵. Current DC vaccine protocolsinclude pre-conditioning the vaccine injection site with inflammatorycytokines or ex vivo stimulation of DCs with TLR ligands andpro-inflammatory cytokines, consisting primarily of MCconstituents53,54. Despite its numerous immunosuppressive functions⁸⁻¹²,PGE₂ has been used for the past few years as an indispensible componentof the DC maturation cocktail because it stimulates the migratorycapacity of DCs by up-regulating CCR7 and sensitization to its ligands.Alternative approaches enhancing DCs migration without PGE₂, should bebeneficial for DC-based vaccine improvement.

The results of our study show that iCD40 signaling not only up-regulatesCCR7 expression on DCs but also stimulates their chemotaxis to CCL19 invitro. Additionally, immature DCs transduced with iCD40 were able tomigrate as efficiently as MC-stimulated DCs both in vitro and in vivo.Moreover, migration of iCD40-DCs was further induced when cells werestimulated with TLR-4 ligands. It was recently shown that stimulation ofCCR7 increases the migratory rate of DCs, indicating that this receptorcan regulate DC locomotion and motility⁵⁵⁻⁵⁷. It has been shown thatstimulation of CCR7 enhances the mature phenotype of DCs58. Thus, bytransduction of DCs with iCD40, CCR7 expression, DC migration andmaturation status have been enhanced, obviating the need for PGE₂.Further studies are underway to identify the specific mechanisms ofiCD40 on DC migration.

Finally, iCD40 stimulation of DCs was capable of inducing a potentcytotoxic T cell response to the prostate-specific antigen, PSMA, whichwas capable of significantly increased killing of target LNCaP cells.Based on these observations, a clinical vaccine trial is planned usingiCD40-transduced, PSMA protein-loaded DCs, in combination with AP20187,in patients with advanced, hormone refractory prostate cancer.Ultimately, by expanding antigen-specific T cells, the DC-based vaccineapproach could compliment recently described techniques that are basedon the expansion of tumor-derived T cells or the genetic modification ofpolyclonal endogenous T cells to antigen-specificity^(59,60).

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Vaccine. 2002; 20 Suppl 4:A8-A22.-   30. Liu J, Cao S, Herman L M, Ma X. Differential regulation of    interleukin (IL)-12 p35 and p40 gene expression and interferon    (IFN)-gamma-primed IL-12 production by IFN regulatory factor 1. J    Exp Med. 2003; 198:1265-1276.-   31. Contin C, Pitard V, Itai T, Nagata S, Moreau J F,    Dechanet-Merville J. Membrane-anchored CD40 is processed by the    tumor necrosis factor-alpha-converting enzyme. Implications for CD40    signaling J Biol Chem. 2003; 278:32801-32809.-   32. Tone M, Tone Y, Fairchild P J, Wykes M, Waldmann H. Regulation    of CD40 function by its isoforms generated through alternative    splicing. Proc Natl Acad Sci USA. 2001; 98:1751-1756.-   33. Wesemann D R, Dong Y, O'Keefe G M, Nguyen V T, Benveniste E N.    Suppressor of cytokine signaling 1 inhibits cytokine induction of    CD40 expression in macrophages. J Immunol. 2002; 169:2354-2360.-   34. Evel-Kabler K, Song X T, Aldrich M, Huang X F, Chen S Y. SOCS1    restricts dendritic cells' ability to break self tolerance and    induce antitumor immunity by regulating IL-12 production and    signaling. J Clin Invest. 2006; 116:90-100.-   35. Pasare C, Medzhitov R. Toll pathway-dependent blockade of    CD4+CD25+ T cell-mediated suppression by dendritic cells. Science.    2003; 299:1033-1036.-   36. Davis M I, Bennett M J, Thomas L M, Bjorkman P J. Crystal    structure of prostate-specific membrane antigen, a tumor marker and    peptidase. Proc Natl Acad Sci USA. 2005; 102:5981-5986.-   37. Cyster J G. Chemokines and cell migration in secondary lymphoid    organs. Science. 1999; 286:2098-2102.-   38. Sanchez-Sanchez N, Riol-Blanco L, Rodriguez-Fernandez J L. The    Multiple Personalities of the Chemokine Receptor CCR7 in Dendritic    Cells. J Immunol. 2006; 176:5153-5159.-   39. De Vries I J, Krooshoop D J, Scharenborg N M, et al. Effective    migration of antigen-pulsed dendritic cells to lymph nodes in    melanoma patients is determined by their maturation state. Cancer    Res. 2003; 63:12-17.-   40. Nestle F O, Banchereau J, Hart D. Dendritic cells: On the move    from bench to bedside. Nat Med. 2001; 7:761-765.-   41. Schuler G, Schuler-Thurner B, Steinman R M. The use of dendritic    cells in cancer immunotherapy. Curr Opin Immunol. 2003; 15:138-147.-   42. Cranmer L D, Trevor K T, Hersh E M. Clinical applications of    dendritic cell vaccination in the treatment of cancer. Cancer    Immunol Immunother. 2004; 53:275-306.-   43. Ridgway D. The first 1000 dendritic cell vaccinees. Cancer    Invest. 2003; 21:873-886.-   44. Dallal R M, Lotze M T. The dendritic cell and human cancer    vaccines. Curr Opin Immunol. 2000; 12:583-588.-   45. Adema G J, de Vries I J, Punt C J, Figdor C G. Migration of    dendritic cell based cancer vaccines: in vivo veritas? Curr Opin    Immunol. 2005; 17:170-174.-   46. Langenkamp A, Messi M, Lanzavecchia A, Sallusto F. Kinetics of    dendritic cell activation: impact on priming of TH1, TH2 and    nonpolarized T cells. Nat Immunol. 2000; 1:311-316.-   47. Napolitani G, Rinaldi A, Bertoni F, Sallusto F, Lanzavecchia A.    Selected Toll-like receptor agonist combinations synergistically    trigger a T helper type 1-polarizing program in dendritic cells. Nat    Immunol. 2005; 6:769-776.-   48. Bisping G, Kropff M, Wenning D, et al. Targeting receptor    kinases by a novel indolinone derivative in multiple myeloma:    abrogation of stroma-derived interleukin-6 secretion and induction    of apoptosis in cytogenetically defined subgroups. Blood. 2006;    107:2079-2089.-   49. Kalams S A, Walker B D. The critical need for CD4 help in    maintaining effective cytotoxic T lymphocyte responses. J Exp Med.    1998; 188:2199-2204.-   50. Kalinski P, Hilkens C M, Snijders A, Snijdewint F G, Kapsenberg    M L. Dendritic cells, obtained from peripheral blood precursors in    the presence of PGE₂, promote Th2 responses. Adv Exp Med Biol. 1997;    417:363-367.-   51. Mcllroy A, Caron G, Blanchard S, et al. Histamine and    prostaglandin E up-regulate the production of Th2-attracting    chemokines (CCL17 and CCL22) and down-regulate IFN-gamma-induced    CXCL10 production by immature human dendritic cells. Immunology.    2006; 117:507-516.-   52. Meyer F, Ramanujam K S, Gobert A P, James S P, Wilson K T.    Cutting edge: cyclooxygenase-2 activation suppresses Th1    polarization in response to Helicobacter pylori. J Immunol. 2003;    171:3913-3917.-   53. Martln-Fontecha A, Sebastiani S, Hopken U E, et al. Regulation    of dendritic cell migration to the draining lymph node: impact on T    lymphocyte traffic and priming. J Exp Med. 2003; 198:615-621.-   54. Prins R M, Craft N, Bruhn K W, et al. The TLR-7 agonist,    imiquimod, enhances dendritic cell survival and promotes tumor    antigen-specific T cell priming: relation to central nervous system    antitumor immunity. J Immunol. 2006; 176:157-164.-   55. Riol-Blanco L, Sanchez-Sanchez N, Torres A, et al. The chemokine    receptor CCR7 activates in dendritic cells two signaling modules    that independently regulate chemotaxis and migratory speed. J    Immunol. 2005; 174:4070-4080.-   56. Palecek S P, Loftus J C, Ginsberg M H, Lauffenburger D A,    Horwitz A F. Integrin-ligand binding properties govern cell    migration speed through cell-substratum adhesiveness. Nature. 1997;    385:537-540.-   57. Yanagawa Y, Onoe K. CCL19 induces rapid dendritic extension of    murine dendritic cells. Blood. 2002; 100:1948-1956.-   58. Marsland B J, Battig P, Bauer M, et al. CCL19 and CCL21 induce a    potent proinflammatory differentiation program in licensed dendritic    cells. Immunity. 2005; 22:493-505.-   59. 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Other documents cited herein are referenced in U.S. patent applicationSer. No. 10/781,384, filed Feb. 18, 2004, entitled “Induced ActivationIn Dendritic Cells,” and naming Spencer et al. as inventors.

Example 8: Inducible Pattern Recognition Receptors

The innate immune system uses several families of pattern recognitionreceptors PRRs to sense pathological infection or injury. One family ofPRRs is the Toll-like receptors (TLRs) that now include about 11 membersin mammals. These typically bind to multi-valent ligands through aleucine-rich motif (LRM). The ligands can come from bacteria, viruses,fungi, or host cells and can bind to TLRs either on the cell surface orwithin endocytic vesicles (especially TLR 3, 7, 8 and 9). Within theircytoplasmic signaling domains, they share a conserved TIR (Toll/IL-1R)domain that binds to downstream TIR-containing adapter molecules, suchas MyD88 and TRIF/TICAM-1, and adapters TIRAM/TICAM-2 and MAL/TIRAP.Additional P RRs include the NOD-like receptors (e.g. NOD1 and NOD2) andthe RIG-like helicases, RIG-I and Mda-5. Many PRRs bind to ligandsthrough flexible LRMs and couple to downstream signaling moleculesthrough protein-protein binding motifs, such as TIR or CARD (caspaserecruitment domain) domains.

Stimulation through TLR-4 in conjunction with signaling through thecostimulatory molecule CD40 can promote high-level maturation andmigratory properties in human monocyte-derived dendritic cells (MoDCs).Based on both published and unpublished data″, this prolonged andenhanced activation state of human MoDCs in vitro and/or in vivo mayboth promote the activation and expansion of autologous tumor-specific Tcells for adoptive immunotherapy and overcome the problems ofself-limiting ex vivo-matured DCs for vaccination.

Currently, there is a limited array of TLR agonists available inclinical studies. So far, the only clinically approved TLR-4 ligand ismonophosphoryl lipid A (MPL). To develop a simplified, unified vectorfor optimal MoDC activation in vitro and in vivo in the absence of addedTLR or costimulatory ligands, a chemical inducer of dimerization(CID)-inducible TLRs (iTLRs) fused to costimulatory receptors, such asCD40, may be developed. Thus, rather than modifying MoDCs with severalactivation reagents, a unified chimeric receptor may be developedcontaining all components necessary for potent, inducible activation ofMoDCs in vitro and in vivo within the context of an immunologicalsynapse. Toward this goal, several iTLRs representing multiple TLRsubfamilies are developed and their efficacy alone and when fused toiCD40 compared. Second, the most potent iCD40-TLR is compared withpreviously identified most potent methods for enhancing DC activation.Lastly synergistic effects are appraised in a spectrum of in vitroassays on human DCs.

Development of synthetic drug-inducible Toll-like receptors andcomposite costimulatory receptors within a single vector for unifiedbroadly applicable immunotherapy. To replace complex, poorly understoodMoDC maturation cocktails or combinations of adjuvants and CD40signaling, CID-inducible versions of toll-like receptor 4 (called iTLR4)and other iTLRs (i.e. TLR3, 7, 8, and 9) are developed and iTLRs areassayed for synergy with iCD40 either in trans or in cis within the samepolypeptide chain. Efficacy is based on induction of transcriptionfactors NF-kB and IRF3/7s, and phosphorylation of p38 and INK in the DCline, 2DSC/1. The most potent inducible receptor is subcloned into anadenovector for efficient transduction of MoDCs.

Comparison of optimum iTLR-CD40 with previously developed approaches(i.e. iCD40, Myr_(F)-ΔAkt, SOCS-1 shRNA) to enhance MoDC activation,survival, and function in vitro and in vivo. The murine DC-vaccinemodels are extended to a preclinical human model for optimizing DCmaturation and activation. Following MoDC modification with the recentlydeveloped “humanized” vectors, DC maturation (e.g. upregulated CD83,CCR7), and survival is compared in the absence of growth factors (i.e.GM-CSF), chemotactic response to CCL19/21 in a 2-chamber assay and invivo in non-myoablatively irradiated scid mice using optical imaging. Inaddition to determining the capacity of enhanced DCs (eDCs) to triggerTh1 polarization (determined via multiplex cytokine assays (e.g. IL-4,IFN-gamma, IL-12, IL-23), and delta-4 mRNA), the activation ofautologous T cells in healthy donors by various eDCs presenting twodistinct cocktails of HLA-A2-restricted antigens, one strong and oneweak is compared. Finally, SOCS-1 depletion is assayed for synergy withiTLR-CD40, iCD40 or Myr_(F)-ΔAkt signaling to produce a furtheroptimized eDC. If synergy is found, a bicistronic adenovector containingboth genetic elements will be developed and characterized. These 2 aimsshould lead to development of an extremely potent DC vaccine platform.

Significance

Dendritic cells Dendritic cells (DCs) play a critical role in initiatingand regulating adaptive immunity^(7,8). Upon detection of “dangersignals”, DCs physiologically adapt to their microenvironment byundergoing a genetic maturation program⁶. Using a broad repertoire ofantigen presentation and costimulatory molecules, DCs are capable ofpotently activating naïve antigen-specific T lymphocytes and regulatingtheir subsequent phenotype and function⁹. In most cases, the developmentof robust cytotoxic T lymphocyte (CU) immunity by DCs requires a“helper” signal from CD4⁺ T cells¹⁰. This signal is comprised of bothsoluble cytokines, such as IL-2, as well as CD40L-mediated stimulationof the surface CD40 receptor on the DC¹¹⁻¹³. A member of the tumornecrosis factor receptor (TNFR) superfamily, CD40 triggers variouspathways within the DC resulting in the upregulation of several antigenpresentation, costimulatory, cytokine, and pro-survival genes, whichcollectively enable the DC to induce CTL activation^(14,15).

Use of DCs in immunotherapy Given the pre-eminent role of DCs asantigen-presenting cells (APCs), their exploitation as natural adjuvantsin vaccination protocols for the treatment of various malignancies isnot surprising^(16,17). Typical applications include harvestingperipheral blood monocytes via leukapheresis, differentiation in culturein GM-CSF and IL-4, and loading immature monocyte (or CD34⁺ precursorcell)-derived DCs (MoDC) with tumor antigens by one of several methods,such as pulsing immature DCs with unfractionated tumor lysates,MHC-eluted peptides, tumor-derived heat shock proteins (HSPs), tumorassociated antigens (TAAs (peptides or proteins)), or transfecting DCswith bulk tumor mRNA, or mRNA coding for TAAs (reviewed in^(18,19)).Antigen-loaded DCs are then typically matured ex vivo with inflammatorycytokines (e.g. TNFalpha, IL 1beta, IL6, and PGE₂) or other adjuvants(e.g. LPS, CpG oligonucleotides) and injected into patients. In eachcase, the immuno-stimulatory properties of the DCs depend on manyvariables, especially the ability to migrate to lymph nodes and fullmaturation status. However, the limited success in recent clinicaltrials with DC immunotherapy has suggested that current protocols needto be refined if DC-based immunotherapy is to be included in thetreatment arsenal alongside more conventional modalities of anti-cancertherapy^(20,21).

Two key limitations of DC-based vaccines are the short lifespan ofmatured DCs and their transient activation state within lymphoidtissues. Less than 24 hours following exposure to lipopolysaccharide(LPS), DCs terminate synthesis of the T_(H)1-polarizing cytokine, IL-12,and become refractory to further stimuli²², limiting their ability toactivate cytotoxic T lymphocytes (CTLs). Other studies indicate that thesurvival of antigen-pulsed DCs within the draining lymph node (LN) islimited to only 48 hours following their delivery, due primarily toelimination by antigen-specific CTLs²³. These findings underscore theneed for improved methods of either prolonging the activation state andlife span of the DCs and/or temporally coordinating the DC activation“window” with engagement of cognate T cells within LNs. Thus, enhancingthe activation and survival of DCs may be critical to promoting immunityagainst tumors.

DC survival DC survival is regulated, at least partly, bypathogen-derived molecules acting through one or more conservedToll-like receptors (TLRs) and T cell-expressed costimulatory molecules(e.g. CD40L and TRANCE), which are partly dependent on Bcl-2 andBcl-x_(L) for anti-apoptotic activity^(3,24-27). Although the importanceof TLR-, CD40-, or Bcl-2-mediated DC longevity has been well documented,homeostatic feedback mechanisms are also likely to limit the utility ofTLR-ligands or Bcl-2 family members to extend DC longevity in tumorvaccine protocols. These include receptor desensitization ordownregulation^(4,28,29), expression of negative regulators forTLR/IL-1Rs, like IRAK-M³⁰ and SOCS-1⁵, and induction of pro-apoptoticmolecules, like Bim³¹, resulting in the neutralization of anti-apoptoticmolecules by TLR signals.

Role of Akt in DC survival Akt/PKB family proteins, major downstreameffectors of PI3 Kinases (PI3K), have been reported as criticalcomponents in the regulation of various biological processes, includinggrowth, survival, transformation, and others (reviewed in 32). In DCs,it has been shown that inhibition of PI3K antagonizes LPS, TRANCE, CD40or PGE₂-mediated dendritic cell survival^(33,34). In addition, recentstudies reveal that some tumors escape from immune surveillance by theinduction of inhibitory molecules, such as ceramide and TGF-beta,resulting in DC apoptosis through the suppression of Akt, NF-kappaB, andBcl-x_(L) ^(35,36). Taken together, these studies suggest that thePI3K/Akt pathway plays an important role in maintaining DC survival;however, the detailed molecular mechanisms have not been fullyaddressed.

Role of CD40 in DC survival and activation Another particularlyattractive target for manipulation is the TNF family receptor, CD40.Unlike pro-inflammatory cytokines or pathogen-associated molecules thatDCs encounter throughout the periphery, the DC-expressed CD40 receptoris engaged by CD4⁺ T helper cells within the LN paracortex via itscognate ligand, CD40L^(12,13,37). Recent studies have further shown thatCD40 stimulation enables DCs to “cross-present” antigen 38 and overcomeperipheral T cell tolerance³⁹, prompting therapeutic studies based onCD40 stimulation. Strategies included systemic delivery of CD40-specificmonoclonal antibodies (mAbs) or of trimerized CD40L⁴⁰, the utilizationof CD40-stimulated, antigen-loaded DC-based vaccines⁴¹, andadministration of genetically modified CD40 ligand (CD40L)-expressingDCs⁴². Despite great potential, several properties of CD40 limit itstherapeutic development, including ubiquitous expression of CD40 by avariety of other cell types, including B cells, macrophages, andendothelial cells¹⁴, increasing the likelihood for side effects due tosystemic administration of CD40 stimuli. Moreover, several mechanismsregulate the surface expression of CD40 by targeting its extracellulardomain, including CD40L-induced cleavage by matrix metalloproteinaseenzymes²⁹, negative feedback degradation by an alternatively splicedCD40 isoform²⁸, and CD40L-mediated endocytosis of CD40.

Therefore, novel DC activation system was developed based on the CD40signaling pathway to extend the pro-stimulatory state of DCs withinlymphoid tissues by providing DC-targeted functionality, temporalcontrol, and resistance to CD40 regulatory mechanisms. This engineeredrecombinant receptor was comprised of the cytoplasmic domain of CD40fused to ligand binding domains and a membrane-targeting sequence (FIG.10 ). Administration of a lipid-permeable, dimerizing drugintraperitoneally led to the potent induction of CD40-dependentsignaling cascades and greatly improved immunogenicity against bothdefined antigens and tumors in vivo relative to other activationmodalities 4. Hence the chimeric CD40 was named inducible CD40 (iCD40).The high utility of iCD40-activated DCs in mice, suggested that methodsto stabilize endogenous CD40 signaling might also enhance the potency ofDC vaccines.

Role of TLRs in DC survival and activation TLRs binds to a variety ofviral and bacterial-derived molecules, which trigger activation oftarget cells, such as T cells, macrophages and dendritic cells. Althoughthe majority of the 10 or so mammalian TLRs utilize a signaling pathwayinitiated by the adapter protein, MyD88, leading to NF-kappaBactivation, TLR3 relies instead on the adapter TRIF, leading to IRF3 andType I interferon induction. Together, these signaling pathways cansynergize to produce high levels of the Th1 cytokine, IL-12⁴³.Interestingly, TLR-4 can utilize both pathways following binding of thepotent mitogen, LPS, or derivatives. Stimulation through TLR-4 inconjunction with signaling through the costimulatory molecule CD40 canpromote high-level maturation and migratory properties in human MoDCs(preliminary data). Currently, there is a limited array of TLR agonistsavailable in clinical studies. So far, the only clinically approvedTLR-4 ligand is monophosphoryl lipid A (MPL).

Like many cell-surface receptors that make a single pass through theplasma membrane, TLRs are likely to all be activated by homo orheterodimerization or oligomerization. Over the past few years therehave been several citations showing homodimerization-mediated activationof TLR-4 and heterodimerization-mediated activation of TLR2 with TLR1and TLR6⁴⁴⁻⁴⁸. Moreover, in a recent article, Ian Wilson and colleaguescrystallized TLR-3 and identified dimerization regions within theextracellular domain, suggesting that it signals as a homodimer followeddsRNA binding⁴⁹. Therefore, it is extremely likely that chemicallyinduced dimerization of TLRs, especially TLR-4, will lead to theirinduction.

Considerations for the development of ex vivo-matured, monocyte-derived“enhanced” human DCs. Our recent published 4 and unpublished (seePreliminary Data) studies have suggested two potent methods to enhanceDC function in vivo, ectopic expression of an optimized, constitutiveAkt (Myr_(F)-ΔAkt) and manipulation of a chimeric inducible CD40 invivo. Complementing this work, Si-Yi Chen (Baylor College of Medicine,Houston, Texas) has shown that lowering SOCS-1 levels in DCs can alsoenhance efficacy⁵. With the addition of inducible TLRs, there would beat least 4 potent methods to activate DCs in vivo. Significantsupporting data in mice for iCD40, Myr_(F)-ΔAkt, and SOCS-1 approaches,and human MoDCs are not identical to murine bone marrow-derived DCs. Inparticular, the most commonly used human DC vaccine protocol involvesdifferentiation of MoDCs from monocytes, prior to treatment with the“gold standard” pro-inflammatory maturation cocktail, containingTNF-alpha, IL-1-beta, IL-6, and PGE₂. Although PGE₂ is considerednecessary to upregulate CCR7 and gain chemotactic responsiveness tolymph node-derived chemokines, CCL19 and CCL21^(50,51), PGE₂ can alsoimpair DC signaling by suppressing bioactive IL12p70 production⁵². Whileit is unlikely that IL12 suppression is permanent in vivo, given theslowly building success rate of DC vaccines⁷, it will be important todetermine prior to clinical applications which of the methods outlinedabove can best overcome PGE₂-mediated IL12 suppression in human MoDCswithout interfering with migratory capacity.

Although clinical success in DC-based vaccines has been modese^(7,20),extremely low side effects and potentially exquisite specificity andsensitivity make this modality attractive. Multiple, potentiallycomplementary approaches to enhance maturation, activity and survival ofantigen-expressing DCs in vivo have been developed. Because interactionwith antigen-specific T cells is likely to be prolonged, these enhancedDCs are likely to improve the clinical outcome of DC vaccines. Thedevelopment of enhanced antigen-expressing DCs not only has potentialapplicability to treating malignancy, but also should be applicable tothe treatment of numerous pathogens, as well. Moreover, this high impactapproach should complement prior efforts by numerous labs, which haveidentified tumor antigens.

Preliminary Studies

The validation for the approach described herein for enhancing DCfunction has been recently described^(4,5). Preliminary data for theeffects of iCD40, Myr_(F)-ΔAkt, and siRNA SOCS-1 follow along with dataon the development of iTLRs.

Characterization of iCD40 functionality in primary DCs and developmentof an iCD40-expressing DC-based prostate cancer vaccine. Afterdemonstrating functionality of iCD40 in murine D2SC/1 cells (⁴ and notshown), which possess many characteristics of freshly isolated DCs,iCD40 functionality in primary bone marrow-derived DCs (BMDCs) byutilizing an iCD40-expressing adenovirus was examined. Ahelper-dependent, ΔE1, ΔE3-type 5 adenoviral vector, named Ad-iCD40-GFP,was engineered to express both iCD40 and EGFP under the control of theCMV early/immediate promoter/enhancer. Ad-iCD40-GFP successfullytransduced and expressed the iCD40 transgene, as well as the EGFPmarker, in purified BMDCs (FIG. 11A,B). Titrating Ad-iCD40-GFP whilemeasuring iCD40-induced upregulation of B7.2 (CD86) showed that maximumdrug-mediated iCD40 activation occurred at around 100 moi and proceededasymptotically to plateau at higher viral titers (data not shown).Although the effects were modest, AP20187 induced the surface expressionof MHC class I K^(b), B7.2, as well as endogenous CD40 oniCD40-expressing BMDCs at 100 moi but not on non-transduced DCs (FIG.11C and not shown). The effects of Ad-iCD40-GFP on BMDCs usingintracellular cytokine staining to evaluate DC expression of theT_(H)1-polarizing cytokine, IL-12 was then investigated. These findingsconfirmed numerous previous reports that an empty adenoviral vector cancontribute to background fluorescence readings by stimulating theproduction of low levels of this cytokine. (FIG. 11D)⁵³. Theseexperiments also revealed that the iCD40 transgene could generate asignificant level of basal signaling at these titers even in the absenceof CID. However, AP20187 exposure of these iCD40-expressing DCs managedto reproducibly overcome these cumulative effects to further increasethe percentage of IL-12⁺ DCs. Interestingly, the stimulation ofIL-12p70/p40 synthesis with LPS and CD40L peaked at 8 hrs and decreasedthereafter, while the percentage of IL-12⁺ DCs continued to increaseuntil at least 24 hrs following Ad-iCD40-GFP transduction. Previous workby Langenkamp et al. has demonstrated that prolonged treatment of DCswith LPS exhausts their capacity for cytokine production⁵⁴. Theseresults imply that the Ad-iCD40-GFP vector, as opposed to the LPS dangersignal, is capable of promoting and maintaining a more durable IL-12response by BMDCs.

In addition to DC activation state, DC longevity is another criticalvariable that influences the generation of T cell-dependent immunity. Infact, CTL-mediated killing of DCs is considered to be a significantmechanism for modulating immune responses while protecting the host fromautoimmune pathologies^(55,56). Other work has established that CD40stimulation of DCs prolongs their survival by a variety of mechanisms,including upregulation of the anti-apoptotic protein bcl-X_(L) and thegranzyme B inhibitor, Spi-6^(57,58). The effects of iCD40 relative toCD40L on DC survival were compared in an in vitro serum-starvationculture assay (FIG. 11E). By analyzing the vital dye (propidium iodide(PI))-positive cell population by flow cytometry, iCD40 expressing-BMDCswere found to exhibit greater longevity under these conditions relativeto non-transduced DCs treated with CD40L. This effect wasiCD40-dependent since Ad-GFP-transduced DCs failed to reflect improvedsurvival under these conditions. This work also showed that exposure ofiCD40 BMDCs to the AP20187 dimerizer drug even further enhanced thissurvival effect relative to untreated BMDCs. Moreover, when Ad-iCD40transduced DCs were CFSE-stained and injected into footpads,significantly increased numbers of DCs were found in popliteal lymphnodes following i.p. injections of AP20187 versus in vitro stimulatediCD40 DCs or LPS/CD40L-treated DCs (FIG. 11F).

Despite well-known Ad-dependent maturation signals and basal signalingeffects of iCD40 in primary BMDCs, enhanced DC activation was detectedin the presence of AP20187. Overall, this data suggests that aninducible CD40 receptor designed to respond to a pharmacological agentis capable of maintaining primary DCs in a sustained state of activationcompared to the more transient effects of CD40L stimulation and thepotentially more complex effects of anti-CD40 antibodies. This data isconsistent with earlier findings describing only short-term DCmodulation for stimuli that target endogenous CD40.

The iCD40 Activation Switch Functions as a Potent Adjuvant forAnti-Tumor DNA Vaccines.

Previous studies have demonstrated that DCs play a critical role in theprocessing and presentation of DNA vaccines to responding T cells⁵⁹. Thein vivo anti-tumor efficacy of iCD40 DC-based vaccines as well as the insitu role of iCD40-expressing DCs in tumor immuno-surveillance was thenstudied. To establish a therapeutic tumor model, C57BL/6 mice wereinoculated s.c. with the EG.7-OVA thymoma tumor line and allowed toprogress until tumor volumes reached approximately 0.5 cm³. Thesetumor-bearing mice were vaccinated with either SIINFEKL-pulsed (SEQ IDNO: 6) wt or iCD40 BMDCs. Vaccination with wt BMDCs, either untreated orstimulated in culture with LPS and CD40L or in vivo with anti-CD40 mAb,failed to slow the overall tumor growth rate (FIG. 12 a ). However, invivo drug-mediated iCD40 activation of BMDC vaccines resulted insustained decreases in tumor size (FIG. 12 b ). In addition, theresponse rate to in vivo activated iCD40-expressing BMDC vaccines wassignificantly higher than the response rates to wild type BMDCs underall other vaccination conditions (70% vs 30%). To confirm theelicitation of tumor antigen-specific T cell responses in tumor-bearingmice, H-2K^(b) OVA₂₅₇₋₂₆₄ tetramer analysis was performed on peripheralblood CD8⁺ T cells. This analysis verified the presence of a expandedpopulation of K^(b) OVA₂₅₇₋₂₆₄-specific CD8⁺ T cells exclusively in micevaccinated with in vivo activated iCD40 BMDCs (FIG. 12 c , data notshown).

Enforced expression of Akt-1 in DCs extended their survival and potency.

Akt-1 is found to be essential for DC survival, especially followinggrowth factor withdrawal. Moreover, overexpression of Akt-1 leads toenhanced DCs (eDCs) with greater apoptosis resistance, increasedmaturation, and improved immunogenicity against highly immunogenictumors. Following is a summary of these results:

Rapid down-regulation of Akt following cytokine withdrawal is preventedby innate and acquired immune signals. To investigate pathways involvedin DC survival following inflammatory stimuli, the initial focus was onthose signaling proteins that were known to be induced by thewell-characterized TLR ligand, LPS, which was previously implicated incell survival. Treatment with PI3 kinase (PI3K) and Src kinaseinhibitors significantly antagonized LPS-mediated survival, whereas JAKand MAPK inhibitors had almost no effect even after 48 hr incubation(data not shown).

To further study the role of PI3K in DC survival, the kinetics of Aktexpression, a key down-stream molecule of PI3K, during GM-CSFdeprivation-mediated DC death⁶⁰ was determined. Interestingly, within 24hours of GM-CSF deprivation, total Akt protein levels were rapidlydown-regulated prior to DC death, mirroring decreases in the proteinlevel of Bcl-2, which has been suggested to be down-regulated uponinduction of DC maturation⁶¹ (not shown). Anti-CD40 mAb and LPS werethen found to protect against GM-CSF deprivation-mediated DC death.Although GM-CSF deprivation consistently down-regulated Akt proteinlevels, LPS or anti-CD40 treatment prevented the down-regulation of Akt.Even though mild manipulation of DCs, such as replating, can contributeto Akt phosphorylation at day 2 following GM-CSF withdrawal, unlikeuntreated control cells, only LPS and CD40 signals maintained relativelyhigh Akt phosphorylation and protein levels on day 4, suggesting thatLPS and CD40 stimulation regulate not only the phosphorylation state butalso the protein level of Akt, thereby promoting DC survival (notshown).

To further test the hypothesis that PI3K and Akt are common regulatorsfor innate and acquired immune signal-mediated DC survival, variousconcentrations (0.05-5 μM) of PI3K inhibitor, wortmannin, in BMDCstreated with LPS or anti-CD40 were tested. Even low wortmanninconcentration (0.05 μM), which has very little effect on other types ofcells (data not shown), rapidly induced DC death in both LPS andanti-CD40 treatment, suggesting that PI3K is a common mediator of DCsurvival by inflammatory stimuli.

Functional role of Akt in LPS mediated-DC survival. To more directlyinvestigate the role of Akt in PI3K-mediated DC survival, a previouslydescribed constitutively active Akt (M-Akt) allele, consisting of fulllength Akt, targeted to intracellular membranes with amyristoylation-targeting sequence from c-Src⁶² was used. However, it hasbeen suggested that c-Src, which is only myristoylated, is excluded fromlipid rafts in fibroblasts because dual acylation, such aspalmitoylation and myristoylation, is important for lipid raftlocalization of Src family kinases⁶³. Therefore, to test the possibilitythat the efficient lipid raft localization of Akt improve its functions,this construct was compared with several distinct deregulated Aktalleles, containing myristoylation-targeting sequences from Src familykinases Src, Fyn and Lck fused to ΔAkt. The pleckstrin homology (PH)domain (residues 1-102) was removed to further improve Akt activity whenPI3K is limiting⁶⁴. Among the 3 membrane-targeting sequences, the Fynmyristoylation-targeting sequence (MF) showed the most efficient lipidraft localization, 2-3-fold NF-κB induction, ˜6-fold induced Akt-S473and GSK3 phosphorylation, and enhanced viability of Jurkat cellsfollowing treatment with PI3K inhibitors (data not shown). Therefore,M_(F)-ΔAkt was used in subsequent experiments. Moreover, for improvedexpression in BMDCs, replication-defective adenovirus, Ad-M_(F)-ΔAkt,expressing functionally optimized Akt was generated. Consistently,Ad-M_(F)-ΔAkt led to higher GSK3α/β phosphorylation in BMDCs, comparedwith Ad-M-Akt⁶². In vitro DC survival assays indicated that both vectorscould significantly inhibit wortmannin-mediated lethality in BMDCsrelative to Ad-GFP (p<0.005). In addition, Ad-M_(F)-ΔAkt moreefficiently protected DCs than Ad-M-Akt. Thus, functionally optimizedAkt almost completely suppresses induction of DC death by PI3K (but notSrc) inhibition.

Akt transduced DCs show prolonged longevity in vitro and in vivo.Previous reports demonstrated a correlation between prolonging DClifespan by overcoming various death signals in lymphoid tissues and theadjuvant potency of DC-based vaccines and T cell dependentimmunity^(4,25,26). Therefore, induction of Akt activity promotes thesurvival of DCs under various conditions was assayed. First, the effectsof Ad-M-Akt and LPS on DC survival following growth factor depravationwas tested. As shown in FIGS. 13 a and 13 b , DCs pre-incubated with LPS(1 μg/ml) or infected with Ad-M-Akt maintained viability at least 5 daysafter GM-CSF withdrawal, whereas DCs untreated or transduced with Ad-GFPunderwent significant cell death by day 4, suggesting that the inductionof Akt inhibits cell death signals mediated by GM-CSF withdrawal invitro.

To further investigate Akt-mediated survival of DCs in vivo, theviability of Ad-M_(F)-ΔAkt-transduced DCs with LPS- or Ad-GFP-transducedDCs in draining lymph nodes was compared. DCs were stained with thefluorescent dye CFSE followed by subcutaneous delivery into the hindlegs of syngeneic mice (FIG. 13 c ). On day 5 after delivery, thequantity of CFSE⁺ M_(F)-ΔAkt-DCs residing in the draining popliteallymph node was ˜1% of total lymph node cells, which was a 2-3-foldhigher percentage than control Ad-GFP-DC-treated mice. Consistent withprevious findings³, the percentage of CFSE⁺ DCs from control miceinjected with untreated or LPS-treated DCs rapidly decreased at latertimepoints, whereas Akt-transduced DCs sustained their disproportionaterepresentation for at least 10 days post-delivery (FIG. 13 d ). Inaddition, the average volume of draining lymph nodes exposed toM_(F)-ΔAkt-DCs was approximately 4-8-fold bigger than that from controlmice, indicating that Ad-M_(F)-ΔAkt transduction strongly enhances thenumber of lymph node resident DCs compared to all negative controlgroups as well as LPS-treated DCs (FIG. 13 e ). Since the arrival ofCFSE⁺ DCs does not seem to differ significantly among the groups in thefirst 24 hr after injection, these data strongly suggest that ex vivotransduction of DCs with Ad-M_(F)-ΔAkt promotes prolonged lifespan,which would result in sustained immunity by overcoming various DC deathsignals in lymphoid tissues.

Akt improves DC ability to induce T cell functions. In addition topromoting DC survival, optimal maturation and DC activation, accompaniedby IL-12 production, is important for naïve T cell priming, leading tocell proliferation and IFN-γ production⁶⁵. To directly test whetherenhanced survival and activation of M_(F)-ΔAkt-DCs can promote T cellfunction, the proliferative response of allogeneic (BALB/c) andsyngeneic OT-1 T cells (expressing transgenic TCRs specific forK^(b)-restricted OVA₂₅₇₋₂₆₄ peptide (SIINFEKL)) (SEQ ID NO: 6) topeptide-pulsed, Akt-transduced DCs was examined. After 24-hr incubationof DCs with syngeneic splenocytes from OT-1 mice, Ad-M_(F)-ΔAkttransduced DCs induced T cell proliferation comparable to LPS-treatedDCs, but higher than Ad-GFP-transduced DCs. However, after ˜72 hrincubation, M_(F)-ΔAkt-DCs induced about two-fold higher T cellproliferation than DCs activated with LPS. Moreover, Ad-M-Akt-transducedDCs also consistently revealed 5-7-fold higher allogeneic T cellproliferation responses than DCs pulsed with LPS or Ad-GFP at lowDC:effector ratios after 72-hr incubations. Furthermore, M_(F)-ΔAkt-DCsproduced at least 7-fold higher IFN-gamma⁺ OVA peptide(SIINFEKL)-specific (SEQ ID NO: 6) splenocytes than DCs treated with LPSor Ad-GFP in our ELISpot assay. Taken together, these data stronglysupport the ability of Akt to induce DC maturation and survival,resulting in robust T cell proliferation and activation.

M_(F)-ΔAkt enhances the DC vaccine ability to eradicate apre-established tumor. To more faithfully reflect clinical applications,antitumor efficacy of M_(F)-ΔAkt-DCs was measured. The induction ofimmunity by Ad-M_(F)-ΔAkt-transduced DC vaccines was monitored afterimmunization of C57BL/6 mice bearing large (˜0.4 cm³) s.c. EG.7-OVAtumors. While control SIINFEKL-pulsed (SEQ ID NO: 6) DC vaccines showedno significant inhibition of tumor growth or increased survival, asingle i.p. dose of peptide-pulsed M_(F)-ΔAkt-DCs led to significanttumor growth inhibition (P<0.05) (FIG. 14 a, b , and data not shown). Atearly timepoints, M_(F)-ΔAkt-DCs successfully suppressed allpre-established EG.7-ova tumors, although 2 of 5 tumors eventuallyrelapsed at later timepoints (data not shown). To measure sustainedantigen-specific T cell responses in tumor bearing mice, H-2K^(b)OVA₂₅₇₋₂₆₄ tetramer analysis was performed on peripheral blood CD8⁺ Tcell harvested 14 days after vaccination. This analysis clearly showedthat the vaccination with Ad-M_(F)-Akt-transduced peptide-pulsed DCs ledto an expanded population of OVA₂₅₇₋₂₆₄ antigen-specific CD8⁺ T cells inmice (FIG. 14 c, d ). These findings indicate a crucial role forincreased longevity of DCs in tumor immunosurveillance and clearlysupport the hypothesis that upregulation of Akt activity in DCs improvesDC function, producing enhanced anti-tumor effects.

Development of CID-inducible TLRs (iTLRs): There are several subgroupsof TLRs based on sublocalization and signaling pathways utilized.Development of both iTLR4, normally localized on the cell surface, andalso iTLR3, 7, 8, and 9, normally localized intracellularly is assayed.Regardless of the normal subcellular localization of the ligand-bindingextracellular domains, the signaling domains are cytoplasmic and shouldsignal properly in all cases if homodimerization is the normal signalingmechanism. Analogous to iCD40, the TLR cytoplasmic signaling domainswere PCR-amplified with flanking XhoI and SalI restriction sites forsubcloning on the 5′ or 3′ side of two chemical inducers of dimerization(CID) binding domains (CBD), FKBP12_(V36) ¹. The chimeric CBD-TLRs werelocalized to the plasma membrane using myristoylation-targeting motifs(FIG. 15 ).

Initial testing of TLRs involved co-transfection of expression vectorsinto Jurkat-TAg or 293 cells along with an NF-kB-responsive SEAP(secreted alkaline phosphatase) reporter plasmid⁶⁶. Interestingly, ourpreliminary data suggested that only iTLR7 and iTLR8 functioned inJurkat-TAg cells, but not iTLR3, 4, and 9, regardless of the relativeposition of the CBDs and TLRs (FIG. 16 and not shown). Additionaltransfections in a panel of cells will be required to determine whetherthis reflects physiological tissue-specific signaling differences orother idiosyncrasies of these chimeric constructs.

Development of aggressive preclinical tumor model for in vivo imaging ofvaccine efficacy. Although subcutaneous tumor models provide aconvenient tool for approximating tumor size, their utility is typicallylimited to non-orthotopic tumors that are reasonably symmetrical. Alsoquantitation of metastasis necessitates euthanasia and is limited to asingle measurement. As an improvement on this mainstay approach, tumorcells were developed that stably express a red-shifted luciferase fromCaribbean click beetles (Pyrophorus plagiophthalamus). Imaging in micefollowing administration of substrate D-Luciferin (FIG. 17 ), confirmseasy detection by either a cooled CCD camera (IVIS™ Imaging System,Xenogen Corp.) or standard calipers. Furthermore, the red-shifted (˜613nM emission) luciferase reporter should permit more linear quantitationof surface distant metastasis.

Research Design and Methods:

Specific Aim 1: Develop synthetic drug-inducible Toll-like receptors andcomposite costimulatory receptors within a single vector for unifiedbroadly applicable immunotherapy.

A. Develop most potent icTLR. This aim attempts to circumvent therequirement for pathogen-derived (or synthetic) adjuvant in DCactivation by combining previous genetic manipulation of DCs with newlydeveloped inducible TLRs. Initially, chimeric iTLR 3,4,7,8, and 9, weredeveloped by cloning the cytoplasmic signaling domains of TLRs 5′(upstream) or 3′ (downstream) of CID-binding domains (FIG. 15 ). Initialscreening in Jurkat-TAG cells revealed that iTLR8 (and to a lesserextent iTLR7) triggered the largest induction of NF-κB (FIG. 16 ).However, the relative strength of various TLRs may be a tissue-specificparameter. To address this, these constructs will first be tested in theDC cell line, D2SC/1 initially with regards to NF-kB activation using anNF-kB SEAP reporter system based on transient transfection of multipleexpression plasmids into target cells. 2DSC/1 cells represent a raresubset of immortalized DC lines that retain both the immature DCphenotype and the ability to mature following activation signals⁶⁷.Since, NF-kB induction is not the only function of TLRs (FIG. 18 ),IRF3/7 induction may also be screened using an interferon(IFN)-stimulated response element (ISRE)-SEAP reporter plasmid thatbinds IRFs and induces reporter activity. To develop ISRE-SEAP, theISRE-containing promoter from ISRE-luc (Stratagene) will replace theSRalpha promoter in our constitutive reporter plasmid pSH1/1(SEAP. As asecondary induction of TLR signaling, JNK and p38 phosphorylation aremonitored by western blotting using phosphorylation-specific antibodies.

Since various distinct TLRs can differentially induce IRF and NF-kB andmay synergize in DC activation and IL-12 production⁴³, initial testingof inducible TLRs, will be followed by combinatorial testing bycotransfection of iTLRs, two-at-a-time. Although both normalhomodimerization and more unpredictable heterodimerization may occur,this approach should reveal synergism between different classes of TLRs.Activation of synergistic TLR pairs should confer enhancedimmunostimulatory capacities to DCs. If synergism can be detected, a newseries of constructs that are comprised of two tandem distinct (oridentical) TLRs, called inducible composite TLRs (icTLRs) are tested(FIG. 19 ). In this case cytoplasmic XhoI-SalI-flanked TLR signalingdomains from above are combined in various arrangements upstream anddownstream of CBDs. Finally, the two most potent constructs are modifiedto contain the cytoplasmic domain of CD40, previously demonstrated to beactivated by CID (FIG. 20 ).

Transfection of DCs. Although transfection of DCs can be problematic, animproved method of electroporation was recently described by Vieweg andcolleagues⁶⁸. In their approach, survival following electroporation (300V, 150 mF (Gene Pulser II: Bio-Rad)) is enhanced by resuspending DCs(4×10⁷/ml) in high potassium ion ViaSpan buffer (Barr Laboratories).Additionally, if transfection efficiency is still too low, expressionvector pRSV-TAg, containing SV40 large T antigen for amplifying our pSH1series expression vectors, which all contain the SV40 origin ofreplication will be cotransfected.

B. Develop adenovector expressing unified activation gene icTLR/CD40.Although D2SC/1 is a useful cell model for preclinical studies, theimmunoregulatory genes will next be assayed in primary mouse and humanDCs prior to clinical applications. To facilitate efficient genetransfer to primary cells, the most potent construct(s) is subclonedinto adenovirus shuttle vector, pShuttle-X or pDNR-CMV and furthertransferred into Ad5 vector, pAdeno-X (BD) or AdXLP (BD), respectively.Preparation of high-titer virus is carried out. As has been achievedwith previously developed Ad5/f35-iCD40, this vector is tested in bothhuman and mouse DCs. Although Ad5/f35 pseudotyped adenovectors improvetransduction efficiency a bit in human DCs, “pure” Ad5 envelopedadenovectors will be used to permit additional transduction of murineDCs.

For human studies, MoDCs are prepared by standard incubation of adherentperipheral blood DC precursors in GM-CSF and IL-4. Immature DCs aretransduced with the developed icTLR/CD40 vector and control vectors(e.g. Ad5/f35-iCD40 and Ad5/f35-EGFP). Standard MoDC assays formaturation and activity are described herein and also include, forexample, flow cytometry analysis of maturation markers (e.g. CD40, CD80,CD86, HLA class I and II, CCR7), IL-12 production, migration, andactivation of antigen-specific T cells.

Expected outcomes, possible pitfalls and alternative experiments: SinceMPL synergizes with iCD40, iTLR4 will likely synergize with iCD40;however, due to the vagaries of protein engineering, placing CD40 andTLR signaling domains in tandem may interfere with the signalingpathways activated by isolated domains. Therefore, if tandem domainshave unanticipated signaling, the constructs will be coexpressed inviral vectors using alternative strategies, such as use of bicistronicexpression cassettes or cloning into the E3 region of ΔE1ΔE3adenovectors. Also, chimeric receptors may not signal identical to theendogenous proteins. Therefore, although TLR4 is thought to be the mostpotent TLR for activation of myeloid DCs, an alternative TLR(s) mayfunction better when converted to a CID-activated receptor. Moreover,synergism between iTLRs and constitutive Akt, M_(F)-ΔAkt, or siRNASOCS-1 may be found to be more potent than iCD40 and iTLR. In thesecases, other combinations of immune regulatory genes may be combined inmulticistronic adenovectors.

Specific Aim 2: Comparison of optimum iTLR-CD40 with previouslydeveloped approaches (i.e. iCD40, Myr_(F)-ΔAkt, SOCS-1 shRNA) to enhanceMoDC activation, survival, and function in vitro and in vivo.

Due to the pivotal role that DCs play in regulating adaptive immunity,there are many homeostatic mechanisms that downregulate DC activity.Nevertheless, heightened activation may be required for overcomingtumor- or viral-derived tolerogenic mechanisms. Several methods tocircumvent these homeostatic mechanisms are discussed herein. InducibleCD40 can be activated in vivo within the context of an immunologicalsynapse and lacks its extracellular domain, bypassing several negativefeedback mechanisms that target this domain. “Optimized”, constitutivelyactive Akt, M_(F)-ΔAkt, is based on lipid-raft targeting of a truncatedAkt1 allele. Reducing the inhibitor SOCS-1 with siRNA technologyincreases toll-receptor signaling and Type I interferon production.Thus, all three methods have the capacity to enhance MoDCs.

Preparation of MoDCs: For most experiments based on optimization ofenhanced DCs (eDCs), monocyte-derived DCs are differentiated andenriched from peripheral blood mononuclear cells obtained from the BloodBank or healthy volunteers. Briefly, DC precursors are isolated bybuoyant density techniques (Histopaque: Sigma-Aldrich) and then adherent(and semi-adherent) cells are cultured for 5 days in serum free X-VIVO15 DC medium (Cambrex Bio Science) in the presence of cytokines GM-CSF(800 U/ml) and IL-4 (500 U/ml) (R&D Systems, Minneapolis, MN). Following5 days in culture, immature DCs are incubated for an additional 24 hoursin the presence of adenovectors expressing iCD40 (i.e. Ad5/f35-iCD40),constitutive Akt (Ad5/f35-M_(F)-ΔAkt), shRNA SOCS1 (Ad5-shSOCS1), orAd5-iTLR/CD40 at 10,000 viral particles (vp) per cell. (Note: Ad5vectors may be added at vp to compensate partly for somewhat reducedtransduction efficiency). In a subset of samples, additional TLR4 ligandmonophosphoryl lipid A (MPL; 1 mg/ml) or dimerizer AP20817 (100 nM;iCD40-DCs only) will be added for complete maturation.

Determination of maturation state of MoDCs: A number of surface proteins(“markers”) are induced during MoDC activation, including CD25, CD40,CD80, CD83, CD86, HLA class I and class II, CCR7 and others. Preliminarystudies demonstrated that iCD40 signaling alone is sufficient toupregulate CD83 and CCR7 on MoDCs (not shown). Additional TLR4 signaling(via MPL) leads to additive (or synergistic) activation of allmaturation markers (FIG. 21 and not shown). Therefore, at a fixed vpnumber, induction of maturation markers (determined by flow cytometry)by all four viral vectors either alone or in combination with MPL isevaluated. Maturation by the previous “gold standard” maturationcocktail (MC), comprised of IL-la, IL-6, TNFa, and PGE₂, acts aspositive control and non-treated (mock) immature DCs serve as negativecontrols in these and the following experiments. In addition tophenotypic analysis of cell surface markers, production of IL-12 andother T_(H)1-polarizing cytokines (e.g. IL-23, TNFa), are also importantfor optimal anti-tumor immunity. While iCD40 is not sufficient for IL-12production, combinations of MPL and iCD40 lead to potent synergisticproduction of IL-12 (FIG. 22 ). Therefore, DC culture supernatants,stimulated as above, are harvested 24 and 48 hours after transductionand maturation. IL-12 p70 levels, IL-12/IL-23 p40 dimers and TNFaconcentrations are determined by colorimetric sandwich ELISA assays (BDBiosciences). Alternatively, multiplex beads developed by BD tosimultaneously assay multiple additional cytokines (e.g. IL-1, IL-6,IFNa, etc.) may be used.

Determination of migration capacity: Unlike murine bone marrow-derivedDCs (BMDCs) that are competent for LN migration, immature MoDCs aredeficient in this crucial function. While PGE₂ is typically used toupregulate CCR7 and migratory capacity, the utility of PGE₂ is temperedby potential deleterious effects, which include down regulation of CD40signaling and IL-12 production and upregulation of IL-10^(50,52,69).Moreover, even in the presence of PGE₂, migration to LNs is modest andaround 1-2% of injected cells⁷⁰. Although CCR7 expression is likely aprerequisite for migration to lymph nodes, chemotactic responsiveness tothe LN-derived CCR7 chemokines, CCL19 and CCL21, is a more directmeasure of likely migration to lymph nodes. Therefore, migration toCCL19/MIP3b may be compared in a modified 2-chamber assay.

Preliminary experiments demonstrate the surprising result that iCD40signaling is sufficient for migratory capacity even in the absence ofPGE₂ (FIG. 23 ). In this assay MoDCs were transduced with Ad5/f35-ihCD40and labeled with fluorescent dye, Green-CMFDA (Molecular Probes). Cellswere placed in the top chamber of a 2-chamber 8-mm assay plate and totalfluorescence in the bottom chamber was quantitated and compared withPGE₂-mediated stimulation. Similarly, the migratory capacity in vitro ofiCD40-TLR-, iCD40-, and Akt-MoDCs, and SOCS1-deficient MoDCsindividually and in combination with and without TLR4 ligands may becompared.

As a second more direct assay for migration capacity, migration in vivomay be compared by injecting eDCs into the lower leg ofnon-myeloablatively irradiated immunodeficient SCID mice. Minimalradiation (˜250 rad) is needed to suppress natural killer (NK) cellactivity against xenogeneic cells. Despite species differences, humanMoDCs can respond to murine chemokines and migrate to draining LNs 71.To visualize successfully migrated MoDCs, cells are labeled with thefluorescent dye, Green-CMFDA cell tracker, which is quantitated by flowcytometry. Second, in addition to adenovector-mediated “enhancement”,MoDCs are transduced with adenovector, Ad5/f35-CBR, expressingred-shifted (510 nm excitation peak) click beetle (Pyrophorusplagiophthalamus) luciferase (Promega). Use of the CBR luciferase alleleshould more easily allow detection of bioluminescent DCs (using ourIVIS® Imaging System (Xenogen Corp, Alameda, CA)) both within thedraining popliteal LN and at more distant and membrane-distal sites.

Activation and polarization of autologous T cells: In addition tomaturation and migration, ability to activate a T_(H)1-biasedantigen-specific immune response in vivo is the sine qua non of DCvaccination against solid tumors. Therefore, the ability of eDCs tostimulate both T helper and cytotoxic function may be evaluated.Initially, stimulation of proliferation of allogeneic CD4″ T cells maybe assayed Enhanced DCs are matured and activated using the conditionsdescribed above, irradiated (3000 rad) and cultured 1:10 with allogeneicmagnetic bead-purified (Miltenyi Biotec, Auburn, CA) CD4⁺ T cells.Proliferation is assessed 4 days later after 16-hour incubation with[³H]-thymidine. To complement these studies, the T_(H)1 polarization(determined by ELISpot assays to IL-4 and IFNg) ability by variousstandard or eDCs may be determined To more specifically assay DCmaturation state, ability to stimulate naïve CTL function is determinedusing HLA-A2-restricted tetramer analysis and CTL assays. (Note: severalHLA-A2 carriers have recently been genotyped). Activation of autologousT cells in healthy donors by various eDCs presenting 2 distinctcocktails of HLA-A2-restricted antigens, one strong and one weak iscompared. CTL assays will be based on antigen-specific lytic activity ofT cells stimulated with standard or eDCs as above. These 4 T cell assaysshould provide a balanced preclinical analysis of enhanced DCs alongwith a functional analysis of the various approaches.

Finally, SOCS-1 depletion will be assessed for synergy with iCD40-TLR,iCD40 or Myr_(F)-ΔAkt signaling to produce a further optimized eDC, andif synergy is found, a bicistronic adenovector containing iCD40 (orMyr_(F)-ΔAkt) and SOCS-1 shRNA may be developed and characterized.

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Nat Med 6, 1154-9 (2000).-   43. Napolitani, G., Rinaldi, A., Bertoni, F., Sallusto, F. &    Lanzavecchia, A. Selected Toll-like receptor agonist combinations    synergistically trigger a T helper type 1-polarizing program in    dendritic cells. Nat Immunol 6, 769-76 (2005).-   44. Medzhitov, R., Preston-Hurlburt, P. & Janeway, C. A., Jr. A    human homologue of the Drosophila Toll protein signals activation of    adaptive immunity. Nature 388, 394-7 (1997).-   45. Hoshino, K. et al. Cutting edge: Toll-like receptor 4    (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide:    evidence for TLR4 as the Lps gene product. J Immunol 162, 3749-52    (1999).-   46. Ozinsky, A. et al. The repertoire for pattern recognition of    pathogens by the innate immune system is defined by cooperation    between toll-like receptors. Proc Natl Acad Sci USA 97, 13766-71    (2000).-   47. Zhang, H., Tay, P. N., Cao, W., Li, W. & Lu, J.    Integrin-nucleated Toll-like receptor (TLR) dimerization reveals    subcellular targeting of TLRs and distinct mechanisms of TLR4    activation and signaling. FEBS Lett 532, 171-6 (2002).-   48. Lee, H. K., Dunzendorfer, S. & Tobias, P. S. Cytoplasmic    domain-mediated dimerizations of toll-like receptor 4 observed by    beta-lactamase enzyme fragment complementation. J Biol Chem 279,    10564-74 (2004).-   49. Choe, J., Kelker, M. S. & Wilson, I. A. Crystal structure of    human toll-like receptor 3 (TLR3) ectodomain. Science 309, 581-5    (2005).-   50. Luft, T. et al. Functionally distinct dendritic cell (DC)    populations induced by physiologic stimuli: prostaglandin E(2)    regulates the migratory capacity of specific DC subsets. Blood 100,    1362-72 (2002).-   51. Scandella, E. et al. CCL19/CCL21-triggered signal transduction    and migration of dendritic cells requires prostaglandin E2. Blood    103, 1595-601 (2004).-   52. Kalinski, P., Vieira, P. L., Schuitemaker, J. H., de Jong, E. C.    & Kapsenberg, M. L. Prostaglandin E(2) is a selective inducer of    interleukin-12 p40 (IL-12p40) production and an inhibitor of    bioactive IL-12p70 heterodimer. Blood 97, 3466-9 (2001).-   53. Korst, R., Mahtabifard, A., Yamada, R., and Crystal, R. Effect    of Adenovirus Gene Transfer Vectors on the Immunologic Functions of    Mouse Dendritic Cells. Molecular Therapy 5, 307-315 (2002).-   54. Langenkamp, A., Messi, M., Lanzavecchia, A., and Sallusto, F.    Kinetics of dendritic cell activation: impact on priming of Th1,    Th2, and nonpolarized T cells. Nature Immunology 1, 311-316 (2000).-   55. Hermans, I., Ritchie, D., Yang, J., Roberts, J., and    Ronchese, F. CD8 T cell-dependent elimination of dendritic cells in    vivo limits the induction of antitumor immunity. Journal of    Immunology 164, 3095-3101 (2000).-   56. Wong, P.a.P., E. Feedback Regulation of Pathogen-Specific T Cell    Priming. Immunity 188, 499-511 (2003).-   57. Miga, A., Masters, S., Durell, B., Gonzalez, M., Jenkins, M.,    Maliszewski, C., Kikutani, H., Wade, W., and Noelle, R. Dendritic    cell longevity and T cell persistence is controlled by CD154-CD40    interactions. European Journal of Immunology 31, 959-965 (2001).-   58. Medema, J., Schuurhuis, D., Rea, D., van Tongeren, J., de Jong,    J., Bres, S., Laban, S., Toes, R., Toebes, M., Schumacher, T.,    Bladergroen, B., Ossendorp, F., Kummer, J., Melief, C., and    Offringa, R. Expression of the serpin serine protease inhibitor 6    protects dendritic cells from cytotoxic T lymphocyte-induced    apoptosis: differential modulation by T helper type 1 and type 2    cells. Journal of Experimental Medicine 194, 657-667 (2001).-   59. Steinman, R.a.P., M. Exploiting dendritic cells to improve    vaccine efficacy. Journal of Clinical Investigation 109, 1519-1526    (2002).-   60. Woltman, A. M. et al. Rapamycin specifically interferes with    GM-CSF signaling in human dendritic cells, leading to apoptosis via    increased p27KIP1 expression. Blood 101, 1439-45 (2003).-   61. Granucci, F. et al. Inducible IL-2 production by dendritic cells    revealed by global gene expression analysis. Nat Immunol 2, 882-8    (2001).-   62. Fujio, Y. & Walsh, K. Akt mediates cytoprotection of endothelial    cells by vascular endothelial growth factor in an    anchorage-dependent manner. J Biol Chem 274, 16349-54 (1999).-   63. Mukherjee, A., Arnaud, L. & Cooper, J. A. Lipid-dependent    recruitment of neuronal Src to lipid rafts in the brain. J Biol Chem    278, 40806-14 (2003).-   64. Li, B., Desai, S. A., MacCorkle-Chosnek, R. A., Fan, L. &    Spencer, D. M. A novel conditional Akt ‘survival switch’ reversibly    protects cells from apoptosis. Gene Ther 9, 233-44. (2002).-   Sporri, R. & Reis e Sousa, C. Inflammatory mediators are    insufficient for full dendritic cell activation and promote    expansion of CD4+ T cell populations lacking helper function. Nat    Immunol 6, 163-70 (2005).-   66. Spencer, D. M., Wandless, T. J., Schreiber, S. L. &    Crabtree, G. R. Controlling signal transduction with synthetic    ligands. Science 262, 1019-1024 (1993).-   67. Granucci, F. et al. Modulation of cytokine expression in mouse    dendritic cell clones. Eur J Immunol 24, 2522-6 (1994).-   68. Su, Z. et al. Telomerase mRNA-transfected dendritic cells    stimulate antigen-specific CD8+ and CD4+ T cell responses in    patients with metastatic prostate cancer. J Immunol 174, 3798-807    (2005).-   69. Scandella, E., Men, Y., Gillessen, S., Forster, R. &    Groettrup, M. Prostaglandin E2 is a key factor for CCR7 surface    expression and migration of monocyte-derived dendritic cells. Blood    100, 1354-61 (2002).-   70. Morse, M. A. et al. Migration of human dendritic cells after    injection in patients with metastatic malignancies. Cancer Res 59,    56-8 (1999).-   71. Hammad, H. et al. Monocyte-derived dendritic cells induce a    house dust mite-specific Th2 allergic inflammation in the lung of    humanized SCID mice: involvement of CCR7. J Immunol 169, 1524-34    (2002).

Example 9: Expression Constructs and Testing

TLRs 3, 4, 7, 8 and 9 were initially selected to construct induciblechimeric proteins as they represent TLRs from the different subfamiliesthat are know to trigger the Th1 cytokine, IL-12, in monocyte-derivedDCs. Further, TLR4 has been shown to trigger signaling following crosslinking of chimeric TLR4 alleles via heterologous extracellular domains.The cytoplasmic domains of each (including TIRs) were PCR-amplified andplaced adjacent (5′ and 3′) to two (2) FKBP12(V36) (F_(v) and F_(v),(wobbled)) genes, which were attached to the plasma membrane using amyristoylation-targeting sequence from c-Src. Chimeric proteins having athird FKBP gene have been developed to improve oligomerization.

Additionally, chimeric versions of adapters MyD88 and TRIF have beengenerated by fusing these cytoplasmic proteins to two (2) FKBPs.Finally, the tandem CARD domains from cytoplasmic PRRs, NOD2 and RIG-I,have been fused to tandem FKBPs. These constructs and reporter assaysare described below.

Constructs:

(i) Inducible iTLRs: TLR3, 4, 7, 8 and 9 were PCR-amplified from cDNAderived from MoDCs. PCR primers were flanked by XhoI and SalIrestriction sites to permit cloning 5′ and 3′ of tandem FKBPs in theXhoI and SalI sites, respectively, of pSH1/M-F_(v′)-F_(vls)-E^(1, 2).The primers used were (a) 5TLR3cX(5′-cgatcactcgagggctggaggatatctttttattgg-3′) (SEQ ID NO: 13) and 3TLR3cS(5′-tgatcggtcgacatgtacagagtttttggatccaagtg-3′) (SEQ ID NO: 14) to givepSH1/M-TLR3-F_(v′)-F_(vls)-E and pSH1/M-F_(v′)-F_(vls)-TLR3-E; (b)5TLR4cX (5′-cgatcactcgagtataagttctattttcacctgatgcttc-3′) (SEQ ID NO: 15)and 3TLR4cS (5′-tgatcggtcgacgatagatgttgcttcctgccaattg-3′) (SEQ ID NO:16) to give pSH1/M-TLR4-F_(v′)-F_(vls)-E andpSH1/M-F_(v′)-F_(vls)-TLR4-E; (c) 5TLR7cS(5′-cgatcagtcgacgatgtgtggtatatttaccatactg-3′) (SEQ ID NO: 17) and3TLR7cS (5′-tgatcggtcgacgaccgtaccttgaacacctgac-3′) (SEQ ID NO: 18) togive pSH1/M-TLR7-F_(v′)-F_(vls)-E and pSH1/M-F_(v′)-F_(vls)-TLR7-E; (d)5TLR8cX (5′-cgatcactcgaggatgtttggtttatatataatgtgtg-3′) (SEQ ID NO: 19)and 3TLR8cS (5′-tcggtcgacgtattgcttaatggaatcgacatac-3′) (SEQ ID NO: 20)to give pSH1/M-TLR8-F_(v′)-F_(vls)-E and pSH1/M-F_(v′)-F_(vls)-TLR8-E;(e) 5TLR9cX (5′-cgatcactcgaggacctctggtactgcttccacc-3′) (SEQ ID NO: 21)and 3TLR9cS (5′-tgatctgtcgacttcggccgtgggtccctggc-3′) (SEQ ID NO: 22) togive pSH1/M-TLR9-F_(v′)-F_(vls)-E and pSH1/M-F_(v′)-F_(vls)-TLR9-E. Allinserts were confirmed by sequencing and for appropriate size by westernblot to the 3′ hemagluttinin (HA) epitope (E). M,myristoylation-targeting sequence from c-Src (residues 1-14). pSH1,expression vector. Additionally, a third XhoI/SalI-linkered F_(v′)domain was added to the XhoI sites of pSH1/M-F_(v′)-F_(vls)-TLR4-E andpSH1/M-F_(v′)-F_(vls)-TLR8-E to get pSH1/M-F_(v′)-F_(vls)-TLR4-E andpSH1/M-F_(v′)-F_(vls)-TLR8-E, respectively, to improve oligomerization.

To faithfully reflect physiological TLR4 signaling, full-length 2.5-kbTLR4 was PCR-amplified from TLR4 cDNA (from the Medzhitov lab) usingSacII and XhoI-linkered primers 5hTLR4(5′-aatctaccgcggccaccatgatgtctgcctcgcgcctg-3′) (SEQ ID NO: 23) and3hTLR4 (5′-tcagttctcgaggatagatgttgcttcctgccaattg-3′) (SEQ ID NO: 24),respectively. The 2546-bp PCR product was subcloned into pCR-Blunt-TOPOand sequenced. The sequence-verified insert was SacII/XhoI-digested andsubcloned into SacII/XhoI digested (and “ClPped”)pSH1/M-F_(v′)-F_(vls)-E to give pSH1/hTLR4-F_(v′)-F_(vls)-E. Anadditional F_(v′) was added to XhoI site to givepSH1/hTLR4-F_(v′)-F_(vls)-E.

(ii) Inducible composite iTLR4-CD40: The 191-bp XhoI-SalI-linkered humanCD40 cytoplasmic domain was PCR-amplified with primers hCD405X(5′-atatactcgagaaaaaggtggccaagaagccaacc-3′) (SEQ ID NO: 25) andhCD403Sns (5′-acatagtcgacctgtctctcctgcactgagatg-3′) (SEQ ID NO: 26) andsubcloned into the SalI site of pSH1/hTLR4-F_(v′)-F_(vls)-E andpSH1/hTLR4-F_(v′)-F_(vls)-E to get pSH1/hTLR4-F_(v′)-F_(vls)-CD40-E andpSH1/hTLR4-F_(v′)-F_(vls)-CD40-E.

(iii) Inducible iNOD2: The ˜800-bp amino terminus of the PRR NOD2(containing tandem CARD domains) was PCR-amplified withXhoI/Sa/I-linkered primers 5NOD2X(5′-atagcactcgagatgggggaagagggtggttcag-3′) (SEQ ID NO: 27) and 3NOD2Sb(5′-cttcatgtcgacgacctccaggacattctctgtg-3′) (SEQ ID NO: 28) and subclonedinto the XhoI and SalI sites of pSH1/S-F_(v′)-F_(vls)-E to givepSH1/S-NOD2-F_(v′)-F_(vls)-E and pSH1/S-F_(v′)-F_(vls)-NOD2-E=Fv′ NOD2.

(iv) Inducible iRIG-I: The ˜650 bp amino terminus of the RNA helicaseRIG-I (containing tandem CARD domains) was PCR-amplified withXhoI/SalI-linkered primers SRIGX(5′-atagcactcgagaccaccgagcagcgacgcag-3′) (SEQ ID NO: 29) and 3RIGS(5′-cttcatgtcgacaatctgtatgtcagaagtttccatc-3′) (SEQ ID NO: 30) andsubcloned into the XhoI and SalI sites of pSH1/S-F_(v′)-F_(vls)-E togive pSH1/S-RIGI-F_(v′)-F_(vls)-E andpSH1/S-F_(v′)-F_(vls)-RIGI-E=Fv′RIG-I.

(v) Inducible iMyD88: Human TIR-containing adapter MyD88 (˜900-bp) wasPCR-amplified from 293 cDNA using XhoI/Sa/I-linkered primers 5MyD88S(5′-acatcaactcgagatggctgcaggaggtcccgg-3′) (SEQ ID NO: 31) and 3MyD88S(5′-actcatagtcgaccagggacaaggccttggcaag-3′) (SEQ ID NO: 32) and subclonedinto the XhoI and SalI sites of pSH1/M-F_(v′)-F_(vls)-E to givepSH1/M-MyD88-F_(v′)-F_(vls)-E and pSH1/M-F_(v′)-F_(vls)-MyD88-E,respectively.

(vi) Inducible iTRIF: Human TIR-containing adapter TRIF2 (˜2150-bp) wasPCR-amplified from 293 cDNA using XhoI/SalI-linkered primers 5TRIFX(5′-acatcaactcgagatggcctgcacaggcccatcac-3′) (SEQ ID NO: 33) and 3TRIFS(5′-actcatagtcgacttctgcctcctgcgtcttgtcc-3′) (SEQ ID NO: 34) andsubcloned into SalI-digested pSH1/M-F_(v′)-F_(vls)-E to givepSH1/M-F_(v′)-F_(vls)-TRIF-E.

(vii) IFN-beta-SEAP: The minimal IFNb

promoter was PCR-amplified from human genomic DNA using primers 5IFNbM1(5′-aactagacgcgtactactaaaatgtaaatgacataggaaaac-3′) (SEQ ID NO: 35) and3IFNbH (5′-gacttgaagcttaacacgaacagtgtcgcctactac-3′) (SEQ ID NO: 36). TheMluI-HindIII-digested fragment was subcloned into a promoter-less SEAPreporter plasmid.

Certain constructs were specifically targeted to plasma membrane lipidrafts using myristoylation sequences from Fyn as well as the PIP2membrane targeting domain of TIRAP.(5)

Secreted alkaline phosphatase (SEAP) assays: Reporters assays wereconducted in human Jurkat-TAg (T cells) or 293 (kidney embryonicepithelial) cells or murine RAW264.7 (macrophage) cells. Jurkat-TAgcells (10⁷) in log-phase growth were electroporated (950 mF, 250 V) with2 mg expression plasmid and 2 mg of reporter plasmid NF-kB-SEAP³ orIFNbeta-TA-SEAP (see above). 293 or RAW264.7 cells (˜2×10⁵ cells per35-mm dish) in log phase were transfected with 6 ml of FuGENE-6 ingrowth media. After 24 hr, transformed cells were stimulated with CID.After an additional 20 h, supernatants were assayed for SEAP activity asdescribed previously³.

Tissue culture: Jurkat-TAg and RAW264.7 cells were grown in RPMI 1640medium, 10% fetal bovine serum (FBS), 10 mM HEPES (pH 7.14), penicillin(100 U/ml) and streptomycin (100 mg/ml). 293 cells were grown inDulbecco's modified Eagle's medium, 10% FBS, and pen-strep.

Western blots analysis: Protein expression was determined by westernblot using antibodies to the common hemagluttinin (HA) epitope (E) tag.

Results

Chimeric iTLR4 with the PIP2 membrane targeting motif is activated2-fold. The construct encoded two ligand binding domains. However, therest of the iTLRs are not induced at robust levels by CID in 293, RAW orD2SC1 cells, as observed in reporter assays (FIGS. 25A and 25B). Thismight be attributed to the varied membrane targeting requirements of theiTLRs. Therefore, inducible Nod2 and RIG-1 were developed, which arecytoplasmic PRRs that do not need targeting to the plasma membrane.While iNod2 was activated 2 fold by the dimerizer drug in 293 cells, nosuch effect is observed in RAW 264.7 cells. With the addition ofincreasing concentrations of CID, iNod2 activity decreases in RAW cells.Also the effect of iNod2 and iCD40 together, on NFkappaB activation, isadditive in 293 cells (FIG. 26 ). iRIG-1 is activated by 2.5 fold (FIG.27 ). Inducible versions of the full-length adaptor molecules MyD88 andTRIF that are the primary mediators of signaling downstream of TLRs arein the screening process.

CITATIONS REFERRED TO IN THIS EXAMPLE

-   1. Xie, X. et al. Adenovirus-mediated tissue-targeted expression of    a caspase-9-based artificial death switch for the treatment of    prostate cancer. Cancer Res 61, 6795-804. (2001).-   2. Fan, L., Freeman, K. W., Khan, T., Pham, E. & Spencer, D. M. in    Human Gene Therapy 2273-2285 (1999).-   3. Spencer, D. M., Wandless, T. J., Schreiber, S. L. &    Crabtree, G. R. Controlling signal transduction with synthetic    ligands. Science 262, 1019-1024 (1993).-   4. Thompson, B. S., P. M. Chilton, J. R. Ward, J. T. Evans,    And T. C. Mitchell. 2005. The Low-Toxicity Versions Of Lps, Mpl    Adjuvant And Rc529, Are Efficient Adjuvants For Cd4+ T Cells. J    Leukoc Biol 78:1273-1280.-   5. Salkowski, C. A., G. R. Detore, And S. N. Vogel. 1997.    Lipopolysaccharide And Monophosphoryl Lipid A Differentially    Regulate Interleukin-12, Gamma Interferon, And Interleukin-10 Mrna    Production In Murine Macrophages. Infect Immun 65:3239-3247.-   6. Beutler, B. 2004. Inferences, Questions And Possibilities In    Toll-Like Receptor Signalling. Nature 430:257-263.-   7. Werts, C., S. E. Girardin, And D. J. Philpott. 2006. Tir, Card    And Pyrin: Three Domains For An Antimicrobial Triad. Cell Death    Differ 13:798-815.-   8. Kagan, J. C., And R. Medzhitov. 2006. Phosphoinositide-Mediated    Adaptor Recruitment Controls Toll-Like Receptor Signaling. Cell    125:943-955.

Example 10: Drug-Dependent Induction of NF-Kappa B Activityin CellsTransfected with iRIG-I, iCD40, and iNOD2

293 cells were transfected with 1 microgram NF-KappaB-SEAP reporterconstruct+1 microgram inducible PRR construct using Fugene 6transfection reagent. The transfections were performed in a 6-well plateat 1*10⁶ cells/well or transfection.

Jurkat TAg cells were transfected with 2 micrograms NF-kappa B-SEAPreporter construct and 3 micrograms inducible PRR construct usingelectroporation at 950 microF and kV. The cells were transfected at10*10⁶ cells/transfection.

24 hours later, the cells were plated in a 96-well plate with 2different concentrations of AP20187 (100 nM and 1000 nM). After afurther 24 hour incubation at 37° C., 5% CO₂, supernatants werecollected and analyzed for SEAP activity by incubation with SEAPsubstrate, 4-methylumbilliferyl phosphate (MUP). Fluorescence wasdetermined at excitation 355 nm and emission 460 nm using a FLUOstarOptima plate reader (BMG Labtech).

For iNOD2 and combination experiments, transfections were normalized fortotal DNA using an “empty” expression vector, pSH1/S-Fv′-Fvls-E.

FIGS. 31-34 are graphs that show drug-dependent induction of NF-kappaBactivity and SEAP reporter counts. Each graph is representative of aseparate individual experiment.

For purposes of clarity in the graphs, some of the vectors were renamedfor the figures.

Fy′RIG-I=pSH1-Fv′Fvls-RIG-I=pSH1/S-Fv′-Fvls-RIG-IFv′NOD2=pSH1-Fv′Fvls-NOD2=pSH1/S-Fv′-Fvls-NOD2-EFv′2NOD2=pSH1-Fv′2Fyls-NOD2Fv′NOD2+=pSH1-Fv′Fvls-NOD2 (SFpk3-NOD2 sequence (Ogura, Y., et al. J.Biol. Chem. 276:4812-18 (2001))Fv′ CD40=pSH1-Fv′Fvls-CD40

Example 11: Examples of Particular Embodiments

Examples of certain non-limiting embodiments of the invention are listedhereafter.

A1. A method for activating an antigen-presenting cell, which comprises:

-   -   (a) transducing an antigen-presenting cell with a nucleic acid        having a nucleotide sequence that encodes a chimeric protein,        wherein the chimeric protein comprises a membrane targeting        region, a ligand-binding region and a CD40 cytoplasmic        polypeptide region lacking the CD40 extracellular domain;    -   (b) contacting the antigen-presenting cell with a non-protein        multimeric ligand that binds to the ligand-binding region; and    -   (c) contacting the antigen-presenting cell with a Pattern        Recognition Receptor (PRR) ligand, whereby the        antigen-presenting cell is activated.

A2. The method of embodiment A1, wherein the membrane targeting regionis a myristoylation targeting region.

A3. The method of embodiment A1, wherein the CD40 cytoplasmicpolypeptide region is encoded by a polynucleotide sequence in SEQ ID NO:1.

A4. The method of embodiment A1, wherein the multimeric ligand is asmall molecule.

A5. The method of embodiment A4, wherein the multimeric ligand isdimeric.

A6. The method of embodiment A5, wherein the multimeric ligand is adimeric FK506 or a dimeric FK506 analog.

A7. The method of embodiment A6, wherein the multimeric ligand isAP1903.

A8. The method of any of embodiments A1-A7, A15-A22, wherein the PRRligand is selected from the group consisting of RIG1 ligand, Mac-1ligand, LRP ligand, peptidoglycan ligand, techoic acid ligand, CD11c/CR4ligand, TLR ligand, PGRP ligand, NOD1 ligand, and NOD2 ligand.

A9. The method of any of embodiments A1-A7, A15-A22, wherein the PRRligand is a Toll like receptor (TLR) ligand.

A10. The method of any of embodiments A1-A7, A15-A22, wherein the PRRligand is RIG1 ligand or NOD2 ligand.

A11. The method of embodiment A9, wherein the TLR ligand is selectedfrom the group consisting of lipopolysaccharide (LPS), MPL, FSL-1, Pam3,CSK4, poly(I:C), synthetic imidazoquinoline resiquimod (R848) and CpG.

A12. The method of embodiment A9, wherein the TLR ligand is a TLR4ligand.

A13. The method of embodiment A12, wherein the TLR ligand islipopolysaccharide (LPS).

A14. The method of embodiment A12 wherein the TLR ligand ismonophosphoryl lipid A (MPL).

A15. The method of embodiment A1, wherein the nucleic acid is containedwithin a viral vector.

A16. The method of embodiment A15, wherein the viral vector is anadenoviral vector.

A17. The method of embodiment A1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo.

A18. The method of embodiment A1, wherein the antigen-presenting cell istransduced with the nucleic acid in vivo.

A19. The method of embodiment A1, wherein the antigen-presenting cell isa dendritic cell.

A20. The method of embodiment A19, wherein the dendritic cell is a humandendritic cell.

A21. The method of embodiment A1, wherein the antigen-presenting cell isnot contacted with prostaglandin E₂ (PGE₂) when contacted with themultimeric ligand.

A22. The method of embodiment A1, wherein the antigen-presenting cell isnot contacted with a composition comprising prostaglandin E₂ (PGE₂) andone or more components selected from the group consisting of IL-1beta,IL-6 and TNF alpha.

B1. A method for activating an antigen-presenting cell, which comprises:

-   -   (a) transducing an antigen-presenting cell with a nucleic acid        having a nucleotide sequence that encodes a chimeric protein,        wherein the chimeric protein comprises a membrane targeting        region, a ligand-binding region and a CD40 cytoplasmic        polypeptide region lacking the CD40 extracellular domain; and    -   (b) contacting the antigen-presenting cell with a non-protein        multimeric ligand that binds to the ligand-binding region,        wherein the antigen-presenting cell is not contacted with        prostaglandin E₂ (PGE₂) when contacted with the multimeric        ligand, whereby the antigen-presenting cell is activated.

B2. The method of embodiment B1, wherein the membrane targeting regionis a myristoylation targeting region.

B3. The method of embodiment B1, wherein the CD40 cytoplasmicpolypeptide region is encoded by a polynucleotide sequence in SEQ ID NO:1.

B4. The method of embodiment B1, wherein the multimeric ligand is asmall molecule.

B5. The method of embodiment B4, wherein the multimeric ligand isdimeric.

B6. The method of embodiment B5, wherein the multimeric ligand is adimeric FK506 or a dimeric FK506 analog.

B7. The method of embodiment B6, wherein the multimeric ligand isAP1903.

B8. The method of any of embodiments B1-B7, B13-B22, which furthercomprises contacting the antigen-presenting cell with a PatternRecognition Receptor (PRR) ligand.

B9. The method of embodiment B8, wherein the PRR ligand is selected fromthe group consisting of RIG1 ligand, Mac-1 ligand, LRP ligand,peptidoglycan ligand, techoic acid ligand, CD11c/CR4 ligand, TLR ligand,PGRP ligand, NOD1 ligand, and NOD2 ligand.

B10. The method of embodiment B8, wherein the PRR ligand is RIG1 ligandor NOD2 ligand.

B11. The method of embodiment B8, wherein the PRR ligand is a TLRligand.

B12. The method of embodiment B11, wherein the TLR ligand is selectedfrom the group consisting of lipopolysaccharide (LPS), MPL, FSL-1, Pam3,CSK4, poly(I:C), synthetic imidazoquinoline resiquimod (R848) and CpG.

B13. The method of embodiment B11, wherein the TLR ligand is a TLR4ligand.

B14. The method of embodiment B12, wherein the TLR ligand islipopolysaccharide (LPS).

B15. The method of embodiment B13, wherein the TLR ligand ismonophosphoryl lipid A (MPL).

B16. The method of embodiment B1, wherein the nucleic acid is containedwithin a viral vector.

B17. The method of embodiment B16, wherein the viral vector is anadenoviral vector.

B18. The method of embodiment B1, wherein the antigen-presenting cell istransduced ex vivo with the nucleic acid.

B19. The method of embodiment B1, wherein the antigen-presenting cell istransduced in vivo with the nucleic acid.

B20. The method of embodiment B1, wherein the antigen-presenting cell isa dendritic cell.

B21. The method of embodiment B20, wherein the dendritic cell is a humandendritic cell.

B22. The method of embodiment B1, wherein the antigen-presenting cell isnot contacted with a composition comprising prostaglandin E₂ (PGE₂) andone or more components selected from the group consisting of IL-1beta,IL-6 and TNF alpha.

C1. A method for inducing a cytotoxic T lymphocyte (CTL) immune responseagainst an antigen, which comprises: contacting an antigen-presentingcell sensitized with an antigen with:

-   -   (a) a multimeric ligand that binds to a chimeric protein in the        cell, wherein the chimeric protein comprises a membrane        targeting region, a ligand-binding region and a CD40 cytoplasmic        polypeptide region lacking the CD40 extracellular domain, and    -   (b) a Pattern Recognition receptor (PRR) ligand; whereby a CTL        immune response is induced against the antigen.

C2. The method of embodiment C1, wherein the membrane targeting regionis a myristoylation targeting region.

C3. The method of embodiment C1, wherein the CD40 cytoplasmicpolypeptide region is encoded by a polynucleotide sequence in SEQ ID NO:1.

C4. The method of embodiment C1, wherein the multimeric ligand is asmall molecule.

C5. The method of embodiment C4, wherein the multimeric ligand isdimeric.

C6. The method of embodiment C5, wherein the multimeric ligand is adimeric FK506 or a dimeric FK506 analog.

C7. The method of embodiment C6, wherein the multimeric ligand isAP1903.

C8. The method of any of embodiments C1-C7, C15-C30, wherein the PRRligand is selected from the group consisting of RIG1 ligand, Mac-1ligand, LRP ligand, peptidoglycan ligand, techoic acid ligand, CD11c/CR4ligand, TLR ligand, PGRP ligand, NOD1 ligand, and NOD2 ligand.

C9. The method of any of embodiments C1-C7, C15-C30, wherein the PRRligand is a Toll like receptor (TLR) ligand.

C10. The method of any of embodiments C1-C7, C15-C30, wherein the PRRligand is RIG1 ligand or NOD2 ligand.

C11. The method of embodiment C1, wherein the TLR ligand is selectedfrom the group consisting of lipopolysaccharide (LPS), MPL, FSL-1, Pam3,CSK4, poly(I:C), synthetic imidazoquinoline resiquimod (R848) and CpG.

C12. The method of embodiment C9, wherein the TLR ligand is a TLR4ligand.

C13. The method of embodiment C12, wherein the TLR ligand islipopolysaccharide (LPS).

C14. The method of embodiment C12, wherein the TLR ligand ismonophosphoryl lipid A (MPL).

C15. The method of embodiment C1, wherein the antigen-presenting cell istransduced ex vivo or in vivo with a nucleic acid that encodes thechimeric protein.

C16. The method of embodiment C15, wherein the antigen-presenting cellis transduced ex vivo with the nucleic acid.

C17. The method of embodiment C15, wherein the nucleic acid is containedwithin a viral vector.

C18. The method of embodiment C16, wherein the viral vector is anadenoviral vector.

C19. The method of embodiment C1, wherein the antigen-presenting cell isa dendritic cell.

C20. The method of embodiment C16, wherein the dendritic cell is a humandendritic cell.

C21. The method of embodiment C1, wherein the antigen-presenting cell isnot contacted with prostaglandin E₂ (PGE₂) when contacted with themultimeric ligand.

C22. The method of embodiment C1, wherein the antigen-presenting cell isnot contacted with a composition comprising prostaglandin E₂ (PGE₂) andone or more components selected from the group consisting of IL-1beta,IL-6 and TNF alpha.

C23. The method of embodiment C1, wherein the antigen-presenting cell issensitized to the antigen at the same time the antigen-presenting cellis contacted with the multimeric ligand.

C24. The method of embodiment C1, wherein the antigen-presenting cell ispre-sensitized to the antigen before the antigen-presenting cell iscontacted with the multimerization ligand.

C25. The method of embodiment C1, wherein the antigen-presenting cell iscontacted with the antigen ex vivo.

C26. The method of embodiment C1, wherein the antigen is a tumorantigen.

C27. The method of embodiment C1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo and administered to the subjectby intradermal administration.

C28. The method of embodiment C1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo and administered to the subjectby subcutaneous administration.

C29. The method of embodiment C1, wherein the CTL immune response isinduced by migration of the antigen-presenting cell to a draining lymphnode.

C30. The method of any of embodiments C1-C29, wherein said antigen is aprostate specific membrane antigen.

D1. A method for inducing an immune response against an antigen, whichcomprises: contacting an antigen-presenting cell sensitized with anantigen with a multmeric ligand that binds to a chimeric protein in thecell, wherein:

-   -   (a) the chimeric protein comprises a membrane targeting region,        a ligand-binding region and a CD40 cytoplasmic polypeptide        region lacking the CD40 extracellular domain, and    -   (b) the antigen-presenting cell is not contacted with        prostaglandin E₂ (PGE₂) when contacted with the multimeric        ligand; whereby an immune response against the antigen is        induced.

D2. The method of embodiment D1, wherein the membrane targeting regionis a myristoylation targeting region.

D3. The method of embodiment D1, wherein the CD40 cytoplasmicpolypeptide region is encoded by a polynucleotide sequence in SEQ ID NO:1.

D4. The method of embodiment D1, wherein the multimeric ligand is asmall molecule.

D5. The method of embodiment D4, wherein the multimeric ligand isdimeric.

D6. The method of embodiment D5, wherein the multimeric ligand is adimeric FK506 or a dimeric FK506 analog.

D7. The method of embodiment D6, wherein the multimeric ligand isAP1903.

D8. The method of any of embodiments D1-D7, D13-D30, which furthercomprises contacting the antigen-presenting cell with a PatternRecognition receptor (PRR) ligand.

D9. The method of embodiment D8, wherein the PRR ligand is selected fromthe group consisting of RIG1 ligand, Mac-1 ligand, LRP ligand,peptidoglycan ligand, techoic acid ligand, CD11c/CR4 ligand, TLR ligand,PGRP ligand, NOD1 ligand, and NOD2 ligand.

D10. The method of embodiment D8, wherein the PRR ligand is RIG1 ligandor NOD2 ligand.

D11. The method of embodiment D11, wherein the PRR ligand is a TLRligand.

D12. The method of embodiment D8, wherein the TLR ligand is selectedfrom the group consisting of lipopolysaccharide (LPS), MPL, FSL-1, Pam3,DSK4, poly(I:D), synthetic imidazoquinoline resiquimod (R848) and DpG.

D13. The method of embodiment D11, wherein the TLR ligand is a TLR4ligand.

D14. The method of embodiment D13, wherein the TLR ligand islipopolysaccharide (LPS).

D15. The method of embodiment D13, wherein the TLR ligand ismonophosphoryl lipid A (MPL).

D16. The method of embodiment D1, wherein the antigen-presenting cell istransduced ex vivo or in vivo with a nucleic acid that encodes thechimeric protein.

D17. The method of embodiment D16, wherein the antigen-presenting cellis transduced ex vivo with the nucleic acid.

D18. The method of embodiment D16, wherein the nucleic acid is containedwithin a viral vector.

D19. The method of embodiment D18, wherein the viral vector is anadenoviral vector.

D20. The method of embodiment D1, wherein the antigen-presenting cell isa dendritic cell.

D21. The method of embodiment D20, wherein the dendritic cell is a humandendritic cell.

D22. The method of embodiment D1, wherein the antigen-presenting cell isnot contacted with a composition comprising prostaglandin E₂ (PGE₂) andone or more components selected from the group consisting of IL-1beta,IL-6 and TNF alpha.

D23. The method of embodiment D1, wherein the antigen-presenting cell issensitized to the antigen at the same time the antigen-presenting cellis contacted with the multimeric ligand.

D24. The method of embodiment D1, wherein the antigen-presenting cell ispre-sensitized to the antigen before the antigen-presenting cell iscontacted with the multimerization ligand.

D25. The method of embodiment D1, wherein the antigen-presenting cell iscontacted with the antigen ex vivo.

D26. The method of embodiment D1, wherein the antigen is a tumorantigen.

D27. The method of embodiment D1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo and administered to the subjectby intradermal administration.

D28. The method of embodiment D1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo and administered to the subjectby subcutaneous administration.

D29. The method of embodiment D1, wherein the immune response is acytotoxic T-lymphocyte (DTL) immune response.

D30. The method of embodiment D29, wherein the DTL immune response isinduced by migration of the antigen-presenting cell to a draining lymphnode.

E1. A method for inducing a cytotoxic T lymphocyte (CTL) immune responseagainst an antigen, which comprises: contacting a humanantigen-presenting cell sensitized with an antigen with:

-   -   (a) a multimeric molecule having two or more regions that bind        to and multimerize native CD40, and    -   (b) a Pattern Recognition Receptor (PRR ligand); whereby a CTL        immune response is induced against the antigen.

E2. The method of embodiment E1, wherein the multimeric molecule is anantibody that binds to an epitope in the CD40 extracellular domain.

E3. The method of embodiment E1, wherein the multimeric molecule is aCD40 ligand.

E4. The method of embodiment E1, wherein the multimeric ligand is asmall molecule.

E5. The method of embodiment E4, wherein the multimeric ligand isdimeric.

E6. The method of embodiment E5, wherein the multimeric ligand is adimeric FK506 or a dimeric FK506 analog.

E7. The method of embodiment E6, wherein the multimeric ligand isAP1903.

E8. The method of any of embodiments E1-E7, E15-E22, wherein the PRRligand is selected from the group consisting of RIG1 ligand, Mac-1ligand, LRP ligand, peptidoglycan ligand, techoic acid ligand, CD11c/CR4ligand, TLR ligand, PGRP ligand, NOD1 ligand, and NOD2 ligand.

E9. The method of any of embodiments E1-E7, E15-E22, wherein the PRRligand is a Toll like receptor (TLR) ligand.

E10. The method of any of embodiments E1-E7, E15-E22, wherein the PRRligand is RIG1 ligand or NOD2 ligand.

E11. The method of embodiment E9, wherein the TLR ligand is selectedfrom the group consisting of lipopolysaccharide (LPS), MPL, FSL-1, Pam3,CSK4, poly(I:C), synthetic imidazoquinoline resiquimod (R848) and CpG.

E12. The method of embodiment E9, wherein the TLR ligand is a TLR4ligand.

E13. The method of embodiment E12, wherein the TLR ligand islipopolysaccharide (LPS).

E14. The method of embodiment E12 wherein the TLR ligand ismonophosphoryl lipid A (MPL).

E15. The method of embodiment E1, wherein the nucleic acid is containedwithin a viral vector.

E16. The method of embodiment E15, wherein the viral vector is anadenoviral vector.

E17. The method of embodiment A1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo.

E18. The method of embodiment A1, wherein the antigen-presenting cell istransduced with the nucleic acid in vivo.

E19. The method of embodiment A1, wherein the antigen-presenting cell isa dendritic cell.

E20. The method of embodiment A19, wherein the dendritic cell is a humandendritic cell.

E21. The method of embodiment A1, wherein the antigen-presenting cell isnot contacted with prostaglandin E₂ (PGE₂) when contacted with themultimeric ligand.

E22. The method of embodiment A1, wherein the antigen-presenting cell isnot contacted with a composition comprising prostaglandin E₂ (PGE₂) andone or more components selected from the group consisting of IL-1beta,IL-6 and TNF alpha.

E23. The method of embodiment E1, wherein the antigen-presenting cell issensitized to the antigen at the same time the antigen-presenting cellis contacted with the multimeric ligand.

E24. The method of embodiment E1, wherein the antigen-presenting cell ispre-sensitized to the antigen before the antigen-presenting cell iscontacted with the multimerization ligand.

E25. The method of embodiment E1, wherein the antigen-presenting cell iscontacted with the antigen ex vivo.

E26. The method of embodiment E1, wherein the antigen is a tumorantigen.

E27. The method of embodiment E1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo and administered to the subjectby intradermal administration.

E28. The method of embodiment E1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo and administered to the subjectby subcutaneous administration.

E29. The method of embodiment E1, wherein the CTL immune response isinduced by migration of the antigen-presenting cell to a draining lymphnode.

E30. The method of any of embodiments E1-E29, wherein said antigen is aprostate specific membrane antigen.

F1. A composition comprising an antigen-presenting cell and a PatternRecognition Receptor (PRR) ligand, wherein:

-   -   the antigen-presenting cell is transduced with a nucleic acid        having a nucleotide sequence that encodes a chimeric protein,        and    -   the chimeric protein comprises a membrane targeting region, a        ligand-binding region and a CD40 cytoplasmic polypeptide region        lacking the CD40 extracellular domain.

F2. The composition of embodiment F1, which further comprises anon-protein multimeric ligand that binds to the ligand-binding region.

F3. The method of embodiment F1, wherein the membrane targeting regionis a myristoylation targeting region.

F4. The method of embodiment F1, wherein the CD40 cytoplasmicpolypeptide region is encoded by a polynucleotide sequence in SEQ ID NO:1.

F5. The method of embodiment F2, wherein the multimeric ligand is asmall molecule.

F6. The method of embodiment F2, wherein the multimeric ligand isdimeric.

F7. The method of embodiment F6, wherein the multimeric ligand is adimeric FK506 or a dimeric FK506 analog.

F8. The method of embodiment F2, wherein the multimeric ligand isAP1903.

F9. The method of any of embodiments F1-F8, F15-F27, wherein the PRRligand is selected from the group consisting of RIG1 ligand, Mac-1ligand, LRP ligand, peptidoglycan ligand, techoic acid ligand, CD11c/CR4ligand, TLR ligand, PGRP ligand, NOD1 ligand, and NOD2 ligand.

F10. The method of any of embodiments F1-F8, F15-F27, wherein the PRRligand is a

Toll like receptor (TLR) ligand.

F11. The method of any of embodiments F1-F8, F15-F27, wherein the PRRligand is RIG1 ligand or NOD2 ligand.

F12. The method of embodiment F10, wherein the TLR ligand is selectedfrom the group consisting of lipopolysaccharide (LPS), MPL, FSL-1, Pam3,CSK4, poly(I:C), synthetic imidazoquinoline resiquimod (R848) and CpG.

F13. The method of embodiment F10, wherein the TLR ligand is a TLR4ligand.

F14. The method of embodiment F13, wherein the TLR ligand islipopolysaccharide (LPS).

F15. The method of embodiment F13 wherein the TLR ligand ismonophosphoryl lipid A (MPL).

F16. The method of embodiment F1, wherein the nucleic acid is containedwithin a viral vector.

F17. The method of embodiment F16, wherein the viral vector is anadenoviral vector.

F18. The method of embodiment F1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo.

F19. The method of embodiment F1, wherein the antigen-presenting cell istransduced with the nucleic acid in vivo.

F20. The method of embodiment F1, wherein the antigen-presenting cell isa dendritic cell.

F21. The method of embodiment F20, wherein the dendritic cell is a humandendritic cell.

F22. The method of embodiment F1, wherein the antigen-presenting cell isnot contacted with prostaglandin E₂ (PGE₂) when contacted with themultimeric ligand.

F23. The method of embodiment F1, wherein the antigen-presenting cell isnot contacted with a composition comprising prostaglandin E₂ (PGE₂) andone or more components selected from the group consisting of IL-1beta,IL-6 and TNF alpha.

F24. The method of embodiment F1, wherein the antigen is a tumorantigen.

F25. The method of embodiment F1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo and administered to the subjectby intradermal administration.

F26. The method of embodiment F1, wherein the antigen-presenting cell istransduced with the nucleic acid ex vivo and administered to the subjectby subcutaneous administration.

F27. The method of any of embodiments F1-F26, wherein said antigen is aprostate specific membrane antigen.

G1. A method for assessing migration of an antigen-presenting cell to alymph node, which comprises:

-   -   (a) injecting into a subject an antigen-presenting cell that        produces a detectable protein, and    -   (b) determining the amount of the detectable protein in the        lymph node of the animal, whereby migration of the        antigen-presenting cell to the lymph node is assessed from the        amount of the detectable protein in the lymph node.

G2. The method of embodiment G1, wherein the animal is a rodent.

G3. The method of embodiment G2, wherein the rodent is a mouse.

G4. The method of embodiment G3, wherein the mouse is an irradiatedmouse.

G5. The method of embodiment G1, wherein the detectable protein is aluciferase protein.

G6. The method of embodiment G5, wherein the luciferase protein is froma chick beetle.

G7. The method of embodiment G6, wherein the chick beetle is Pyrophorusplagiophalamus.

G8. The method of embodiment G1, wherein the antigen-presenting cell hasbeen transduced with a nucleic acid having a polynucleotide sequencethat encodes the detectable protein.

G9. The method of embodiment G6, wherein the amount of the luciferaseprotein is determined by administering D-Luciferin to the animal anddetecting the D-Luciferin product generated by the luciferase.

G10. The method of embodiment G1, wherein the lymph node is thepopliteal lymph node.

G11. The method of embodiment G1, wherein the lymph node is the inguinallymph node.

G12. The method of embodiment G1, wherein the antigen-presenting cell isa dendritic cell.

G13. The method of embodiment G12, wherein the dendritic cell is a humandendritic cell.

G14. The method of embodiment G1, wherein the lymph node is removed fromthe animal before the amount of detectable protein is determined.

G15. The method of embodiment G5, wherein the luciferase is ared-shifted luciferase protein.

H1. A method for activating an antigen-presenting cell, which comprises:

-   -   transducing an antigen-presenting cell with a nucleic acid        having a nucleotide sequence that encodes a chimeric protein,        wherein the chimeric protein comprises (i) a membrane targeting        region, (ii) a ligand-binding region and (iii-a) a signaling        region and/or cytoplasmic region of a pattern recognition        receptor (PRR) or (iii-b) an adapter of a PRR; and    -   contacting the antigen-presenting cell with a non-protein        multimeric ligand that binds to the ligand-binding region;    -   whereby the antigen-presenting cell is activated.

H2. The method of embodiment H1, wherein the chimeric protein comprisesa CD40 cytoplasmic polypeptide region lacking the CD40 extracellulardomain.

H2.1. The method of embodiment H1 or H2, wherein the chimeric proteincomprises a signaling region and/or cytoplasmic region of a PRR.

H3. The method of embodiment H2.1, wherein the PRR is a NOD-like PRR.

H4. The method of embodiment H3, wherein the NOD-like PRR is a NOD1 PRR.

H5. The method of embodiment H3, wherein the NOD-like PRR is a NOD2 PRR.

H6. The method of embodiment H1 or H2, wherein the PRR is not a NOD-likePRR.

H7. The method of embodiment H6, wherein the NOD-like PRR is not a NOD1PRR.

H8. The method of embodiment H6, wherein the NOD-like PRR is not a NOD2PRR.

H9. The method of any one of embodiments H1-H3 and H6-H8, wherein thePRR is a RIG-like helicase (RLH).

H10. The method of embodiment H9, wherein the RLH is a RIG-I PRR.

H11. The method of embodiment H9, wherein the RLH is a Mda-5 PRR.

H12. The method of any one of embodiments H1-H3 and H6-H8, wherein thePRR is a Toll-like receptor (TLR) PRR.

H13. The method of embodiment H12, wherein the TLR is selected from thegroup consisting of TLR3, TLR4, TLR7, TLR8 and TLR9.

H14. The method of embodiment H13, wherein the TLR is a TLR4.

H15. The method of embodiment H13, wherein the TLR is a TLR8.

H16. The method of embodiment H13, wherein the TLR is a TLR9.

H17. The method of any one of embodiments H12-H14, wherein the chimericprotein comprises a cytoplasmic region from a TLR PRR.

H18. The method of embodiment H17, wherein the chimeric proteincomprises a TIR domain.

H19. The method of embodiment H18, wherein the chimeric protein consistsessentially of a TIR domain.

H20. The method of embodiment H1 or H2, wherein the chimeric proteincomprises an adapter that binds to a PRR of any one of embodimentsH2-H14.

H21. The method of embodiment H20, wherein the adaptor is selected fromthe group consisting of MyD88, TRIF/TICAM-1, TIRAM/ICAM-2, MAL/TIRAP,TIR and CARD.

H22. The method of any one of embodiments H1-H21, wherein the membranetargeting region is a myristoylation targeting region.

H23. The method of embodiment H2, wherein the CD40 cytoplasmicpolypeptide region is encoded by a polynucleotide sequence in SEQ ID NO:1.

H24. The method of any one of embodiments H1-H23, wherein the ligand isa small molecule.

H25. The method of any one of embodiments H1-H24, wherein the ligand isdimeric.

H26. The method of embodiment H25, wherein the ligand is a dimeric FK506or a dimeric FK506 analog.

H27. The method of embodiment H26, wherein the ligand is AP1903.

H28. The method of any one of embodiments H1-H27, wherein the nucleicacid is contained within a viral vector.

H29. The method of embodiment H28, wherein the viral vector is anadenoviral vector.

H30. The method of any one of embodiments H1-H29, wherein theantigen-presenting cell is contacted with an antigen.

H31. The method of embodiment H30, wherein the antigen-presenting cellis contacted with the antigen ex vivo.

H32. The method of embodiment H30 or H31, wherein the antigen-presentingcell is in a subject and an immune response is generated against theantigen.

H33. The method of embodiment H32, wherein the immune response is acytotoxic T-lymphocyte (CTL) immune response.

H34. The method of embodiment H32 or H33, wherein the immune response isgenerated against a tumor antigen.

H35. The method of any one of embodiments H1-H34, wherein theantigen-presenting cell is transduced with the nucleic acid ex vivo andadministered to the subject by intradermal administration.

H36. The method of any one of embodiments H1-H34, wherein theantigen-presenting cell is transduced with the nucleic acid ex vivo andadministered to the subject by subcutaneous administration.

H37. The method of any one of embodiments H1-H34, wherein theantigen-presenting cell is transduced with the nucleic acid ex vivo.

H38. The method of any one of embodiments H1-H30, wherein theantigen-presenting cell is transduced with the nucleic acid in vivo.

H39. The method of any one of embodiments H1-H38, wherein theantigen-presenting cell is a dendritic cell.

H40. The method of any one of embodiments H1-H39, wherein the nucleicacid comprises a promoter sequence operably linked to the polynucleotidesequence.

I1. A composition which comprises a nucleic acid having a polynucleotidesequence that encodes a chimeric protein, wherein the chimeric proteincomprises (i) a membrane targeting region, (ii) a ligand-binding regionthat binds to a multimeric non-protein ligand, and (iii-a) a signalingregion and/or cytoplasmic region of a pattern recognition receptor (PRR)or (iii-b) an adapter of a PRR.

I2. The composition of embodiment I1, wherein the chimeric proteincomprises a CD40 cytoplasmic polypeptide region lacking the CD40extracellular domain.

I2.1. The method of embodiment I1 or 12, wherein the chimeric proteincomprises a signaling region and/or cytoplasmic region of a PRR.

I3. The composition of embodiment I2.1, wherein the PRR is a NOD-likePRR.

I4. The composition of embodiment I3, wherein the NOD-like PRR is a NOD1PRR.

I5. The composition of embodiment I3, wherein the NOD-like PRR is a NOD2PRR.

I6. The composition of embodiment I1 or I2, wherein the PRR is not aNOD-like PRR.

I7. The composition of embodiment B6, wherein the NOD-like PRR is not aNOD1 PRR.

I8. The composition of embodiment B6, wherein the NOD-like PRR is not aNOD2 PRR.

I9. The composition of any one of embodiments I1-I3 and B6-B8, whereinthe PRR is a RIG-like helicase (RLH).

I10. The composition of embodiment B9, wherein the RLH is a RIG-I PRR.

I11. The composition of embodiment B9, wherein the RLH is a Mda-5 PRR.

I12. The composition of any one of embodiments I1-I3 and B6-B8, whereinthe PRR is a Toll-like receptor (TLR) PRR.

I13. The composition of embodiment I12, wherein the TLR is selected fromthe group consisting of TLR3, TLR4, TLR7, TLR8 and TLR9.

I14. The composition of embodiment I13, wherein the TLR is a TLR4.

I15. The composition of embodiment I13, wherein the TLR is a TLR3.

I16. The composition of embodiment I13, wherein the TLR is a TLR7.

I17. The composition of any one of embodiments I12-I14, wherein thechimeric protein comprises a cytoplasmic region from a TLR PRR.

I18. The composition of embodiment I17, wherein the chimeric proteincomprises a TIR domain.

I19. The composition of embodiment I18, wherein the chimeric proteinconsists essentially of a TIR domain.

I20. The composition of embodiment I1 or I2, wherein the chimericprotein comprises an adapter binds to a PRR of any one of embodimentsI2-I14.

I21. The composition of embodiment I20, wherein the adaptor is selectedfrom the group consisting of MyD88, TRIF/TICAM-1, TIRAM/ICAM-2,MAL/TIRAP, TIR and CARD.

I22. The composition of any one of embodiments I1-I21, wherein themembrane targeting region is a myristoylation targeting region.

I23. The composition of embodiment 12, wherein the CD40 cytoplasmicpolypeptide region is encoded by a polynucleotide sequence in SEQ ID NO:1.

I24. The composition of embodiment I1, wherein the membrane targetingregion is a myristoylation targeting region.

I25. The composition of embodiment 12, wherein the CD40 cytoplasmicpolypeptide region is encoded by a polynucleotide sequence in SEQ ID NO:1.

I26. The composition of any one of embodiments I1-I25, wherein theligand is a small molecule.

I27. The composition of embodiment I26, wherein the ligand is dimeric.

I28. The composition of embodiment I27, wherein the ligand is a dimericFK506 or a dimeric FK506 analog.

I29. The composition of embodiment I28, wherein the ligand is AP1903.

I30. The composition of any one of embodiments I1-I29, wherein thenucleic acid is contained within a viral vector.

I31. The composition of embodiment I30, wherein the viral vector is anadenoviral vector.

I32. The composition of any one of embodiments I1-I31, wherein thenucleic acid comprises a promoter sequence operably linked to thepolynucleotide sequence.

Example 12: Examples of Particular Nucleic Acid and Amino Acid Sequences

(nucleic acid sequence encoding human CD40; Genbank accession no. NM_001250)SEQ ID NO: 1 1gccaaggctg gggcagggga gtcagcagag gcctcgctcg ggcgcccagt ggtcctgccg 61cctggtctca cctcgctatg gttcgtctgc ctctgcagtg cgtcctctgg ggctgcttgc 121tgaccgctgt ccatccagaa ccacccactg catgcagaga aaaacagtac ctaataaaca 181gtcagtgctg ttctttgtgc cagccaggac agaaactggt gagtgactgc acagagttca 241ctgaaacgga atgccttcct tgcggtgaaa gcgaattcct agacacctgg aacagagaga 301cacactgcca ccagcacaaa tactgcgacc ccaacctagg gcttcgggtc cagcagaagg 361gcacctcaga aacagacacc atctgcacct gtgaagaagg ctggcactgt acgagtgagg 421cctgtgagag ctgtgtcctg caccgctcat gctcgcccgg ctttggggtc aagcagattg 481ctacaggggt ttctgatacc atctgcgagc cctgcccagt cggcttcttc tccaatgtgt 541catctgcttt cgaaaaatgt cacccttgga caagctgtga gaccaaagac ctggttgtgc 601aacaggcagg cacaaacaag actgatgttg tctgtggtcc ccaggatcgg ctgagagccc 661tggtggtgat ccccatcatc ttcgggatcc tgtttgccat cctcttggtg ctggtcttta 721tcaaaaaggt ggccaagaag ccaaccaata aggcccccca ccccaagcag gaaccccagg 781agatcaattt tcccgacgat cttcctggct ccaacactgc tgctccagtg caggagactt 841tacatggatg ccaaccggtc acccaggagg atggcaaaga gagtcgcatc tcagtgcagg 901agagacagtg aggctgcacc cacccaggag tgtggccacg tgggcaaaca ggcagttggc 961cagagagcct ggtgctgctg ctgctgtggc gtgagggtga ggggctggca ctgactgggc 1021atagctcccc gcttctgcct gcacccctgc agtttgagac aggagacctg gcactggatg 1081cagaaacagt tcaccttgaa gaacctctca cttcaccctg gagcccatcc agtctcccaa 1141cttgtattaa agacagaggc agaagtttgg tggtggtggt gttggggtat ggtttagtaa 1201tatccaccag accttccgat ccagcagttt ggtgcccaga gaggcatcat ggtggcttcc 1261ctgcgcccag gaagccatat acacagatgc ccattgcagc attgtttgtg atagtgaaca 1321actggaagct gcttaactgt ccatcagcag gagactggct aaataaaatt agaatatatt 1381tatacaacag aatctcaaaa acactgttga gtaaggaaaa aaaggcatgc tgctgaatga 1441tgggtatgga actttttaaa aaagtacatg cttttatgta tgtatattgc ctatggatat 1501atgtataaat acaatatgca tcatatattg atataacaag ggttctggaa gggtacacag 1561aaaacccaca gctcgaagag tggtgacgtc tggggtgggg aagaagggtc tggggg(amino acid sequence encoding human CD40) SEQ ID NO: 2MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ(nucleotide sequence encoding PSMA) SEQ ID NO: 3gcggatccgcatcatcatcatcatcacagctccggaatcgagggacgtggtaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctagcacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctagccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaagcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcctaagcggccgcatagca(PSMA amino acid sequence encoded by SEQ ID NO: 3) SEQ ID NO: 4MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSKHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents. Each of U.S. patent application Ser. No.10/781,384 filed Feb. 18, 2004 and published as US2004/0209836 on Oct.21, 2004, entitled “Induced Activation in Dendritic Cells,” and U.S.Provisional Application No. 60/803,025 filed May 23, 2006, entitled“Modified Dendritic Cells having Enhanced Survival and Immunogenicityand Related Compositions and Methods” is incorporated by referenceherein in its entirety.

Modifications may be made to the foregoing without departing from thebasic aspects of the invention. Although the invention has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, and yet these modifications and improvements are within thescope and spirit of the invention. The invention illustrativelydescribed herein suitably may be practiced in the absence of anyelement(s) not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. Thus, the terms and expressions which have been employed are usedas terms of description and not of limitation, equivalents of thefeatures shown and described, or portions thereof, are not excluded, andit is recognized that various modifications are possible within thescope of the invention. Embodiments of the invention are set forth inthe following embodiments.

What is claimed is:
 1. A composition which comprises a nucleic acidhaving a polynucleotide sequence that encodes a chimeric protein,wherein the chimeric protein comprises (i) a membrane targeting region,(ii) a ligand-binding region that binds to a multimeric non-proteinligand, and (iii-a) a signaling region and/or cytoplasmic region of apattern recognition receptor (PRR) or (iii-b) an adapter of a PRR. 2.The composition of claim 1, wherein the chimeric protein comprises aCD40 cytoplasmic polypeptide region lacking the CD40 extracellulardomain.
 3. The composition of claim 1, wherein the chimeric proteincomprises a signaling region and/or cytoplasmic region of a PRR.
 4. Thecomposition of claim 1, wherein the PRR is a NOD-like PRR.
 5. Thecomposition of claim 4, wherein the NOD-like PRR is a NOD1 PRR.
 6. Thecomposition of claim 4, wherein the NOD-like PRR is a NOD2 PRR.
 7. Thecomposition of claim 1, wherein the PRR is a RIG-like helicase.
 8. Thecomposition of claim 7, wherein the RIG-like helicase is a RIG-I PRR. 9.The composition of claim 1, wherein the PRR is a Toll-like receptor(TLR) PRR.
 10. The composition of claim 1, wherein the chimeric proteincomprises a cytoplasmic region from a TLR PRR.
 11. The composition ofclaim 10, wherein the chimeric protein comprises a TIR domain.
 12. Thecomposition of claim 10, wherein the chimeric protein consistsessentially of a TIR domain.
 13. The composition of claim 1, wherein thechimeric protein comprises an adapter that binds to a PRR.
 14. Thecomposition of claim 2, wherein the chimeric protein comprises anadapter that binds to a PRR.
 15. The composition of claim 13, whereinthe adaptor is selected from the group consisting of MyD88,TRIF/TICAM-1, TIRAM/ICAM-2, MAL/TIRAP, TIR and CARD.
 16. The compositionof claim 1, wherein the membrane targeting region is a myristoylationtargeting region.
 17. The composition of claim 2, wherein the CD40cytoplasmic polypeptide region is encoded by a polynucleotide sequencein SEQ ID NO:
 1. 18. The composition of claim 1, wherein the ligand is asmall molecule.
 19. The composition of claim 18, wherein the ligand is adimeric FK506 or a dimeric FK506 analog.
 20. The composition of claim18, wherein the ligand is AP1903.
 21. The composition of claim 1,wherein the nucleic acid is contained within a viral vector.
 22. Thecomposition of claim 21, wherein the viral vector is an adenoviralvector.
 23. The composition of claim 1, wherein the nucleic acidcomprises a promoter sequence operably linked to the polynucleotidesequence.